Modified Fc proteins comprising site-specific non-natural amino acid residues, conjugates of the same, methods of their preparation and methods of their use

ABSTRACT

Provided herein are modified Fc proteins comprising non-natural amino acid residues at site-specific positions, conjugates of the modified Fc proteins for therapy or diagnosis, compositions comprising the modified Fc proteins and conjugates thereof, methods of their production and methods of their use. The modified Fc proteins and conjugates are useful for methods of treatment and prevention, methods of detection and methods of diagnosis.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit under 35 U.S.C. 119(e) of U.S.Provisional Application No. 61/664,686, filed Jun. 26, 2012, and U.S.Provisional Application No. 61/725,439, filed Nov. 12, 2012. Thecontents of each of the foregoing applications are incorporated hereinby reference in their entireties.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted in ASCII format via EFS-Web and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Sep. 26, 2013, isnamed 108843.00011_SL2.txt and is 17,225 bytes in size.

FIELD

Provided herein are modified Fc proteins comprising non-natural aminoacid residues at site-specific positions, compositions comprising themodified Fc proteins and conjugates thereof, methods of their productionand methods of their use.

BACKGROUND

Antibodies or immunoglobulins comprise two functionally independentparts, a variable domain known as “Fab,” which binds antigen, and aconstant domain known as “Fc,” which links to such effector functions ascomplement activation and attack by phagocytic cells. An immunoglobulinFc domain has a long serum half-life, whereas a Fab domain isshort-lived. See, for example, Capon et al., 1989, Nature 337: 525-531,which is hereby incorporated by reference herein in its entirety.

Immunoglobulin Fc domains and fragments thereof have found widespreaduse as carrier or conjugate proteins for a variety of therapeutic anddiagnostic molecules. When constructed together with, for example, atherapeutic protein or peptide, an immunoglobulin Fc domain can providelonger half-life, or can incorporate such functions as Fc receptorbinding, protein A binding, complement fixation and perhaps evenplacental transfer. As a carrier or conjugate, an Fc domain or fragmentcan be superior to other conjugates, e.g., albumin and PEG: an Fc domainor fragment provides more stability, longer half-life, and reducedimmunogenicity to the molecules attached thereto. For example,attachment of a drug to an Fc domain can increase the serum half-life ofthe drug and reduce the risk of inducing immune responses.

Various methods have been used to attach therapeutic and/or diagnosticmolecules to an Fc domain or fragment. For example, conventionalapproaches for chemical conjugation to the immunoglobulin Fc domaininclude random coupling to naturally occurring primary amines such aslysine and the amino-terminus or carboxylic acids such as glutamic acid,aspartic acid and the carboxy terminus. Alternatively, semi-selectivesite-specific coupling may be achieved through N-terminal conjugationunder appropriate conditions, or derivatized carbohydrates as found onFc proteins isolated from eukaryotic sources, or by partial reductionand coupling of native cysteine residues. (E.g., Kim et al., Apharmaceutical composition comprising an immunoglobulin Fc region as acarrier, WO 2005/047337). While each of these approaches has beenapplied successfully, they typically suffer from varying degrees ofconjugate heterogeneity, relatively low yields and sometimes,significant losses in functional activity.

In addition, modifications have been made to Fc domains and/or fragmentsto optimize their function as carrier or conjugate proteins. Forexample, numerous fusions of proteins and peptides have been engineeredat either the amino- or carboxy-terminus of an Fc domain and/or fragmentthereof. Also, a variety of enzymes and synthetic reporter moleculeshave been chemically conjugated to the side chains of non-terminal aminoacids as well as the derivatized carbohydrate moieties of the Fc domain.Further, polymers such as polyethylene glycol (PEG) have been conjugatedto the Fc domain for the purpose of improved half-life in vivo andreduced immunogenicity.

However, there are problems associated with existing Fc-basedconjugates, including adverse or less optimal effects on thespecificity, efficiency, yield, solubility, and activity of thetherapeutic or diagnostic molecules. There is a need for better Fc-basedcarrier proteins to further improve the properties of the therapeutic ordiagnostic molecules conjugated thereto; in particular, to furtherincrease their half-life in serum.

SUMMARY

Provided herein are modified Fc proteins modified at one or moresite-specific positions with one or more non-natural amino acidresidues. These site-specific positions are optimal for substitution ofa natural amino acid residue with a non-natural amino acid residue. Incertain embodiments, substitution at these site-specific positionsyields Fc proteins that are uniform in substitution, i.e. that aresubstantially modified in the selected position. In certain embodiments,a modified Fc protein substituted at one or more of these site-specificpositions has advantageous production yield, advantageous solubility,advantageous binding and/or advantageous activity. The properties ofthese modified Fc proteins are described in detail in the sectionsbelow.

In one aspect, provided herein are Fc proteins comprising a polypeptidechain having at least one non-natural amino acid residue at a positionin the polypeptide chain that is optimally substitutable. The modifiedFc protein can be a monomer or dimer. Said dimers can be homodimers orheterodimers. The position in the polypeptide chain that is optimallysubstitutable is any position in the polypeptide chain that can providea substitution with optimal yield, uniformity, solubility, bindingand/or activity. The sections below describe in detail the optimallysubstitutable positions of such polypeptide chains. Also described beloware useful Fc proteins containing useful non-natural amino acids.

In a further aspect, provide herein are conjugates of the Fc proteinswith one or more payload molecules. The payload molecule can be anymolecule deemed useful for conjugating to a modified Fc protein. Incertain embodiments, the payload molecule can be a therapeutic moleculeor a diagnostic molecule. The payload molecule can be linked to the Fcprotein directly via a covalent bond or indirectly via a linker.Advantageously, in certain embodiments, the non-natural amino acids ofthe modified Fc proteins provide sites useful for linking to the linkeror to the payload molecule. Accordingly, provided herein are conjugatescomprising a modified Fc protein linked to a payload moiety through anon-natural amino acid at an optimally substitutable site of the Fcprotein.

In another aspect, provided herein are compositions comprising saidmodified Fc proteins or conjugates thereof. Advantageously, suchcompositions can have high uniformity because of the uniformity of thesubstitution of the modified Fc proteins provided herein. In certainembodiments, the compositions comprise a substantial amount of themodified Fc protein or conjugate thereof when measured by total weightof protein or when measured by total weight of Fc protein or conjugate.In certain embodiments, the compositions comprise at least 80% of themodified Fc protein or conjugate thereof, at least 85% of the Fc proteinor conjugate, at least 90% of the modified Fc protein or conjugatethereof, or at least 95% of the Fc protein or conjugate by weight.

In another aspect, provided herein are methods of making the modified Fcproteins. The modified Fc proteins can be made by any technique apparentto those of skill in the art for incorporating non-natural amino acidsinto site-specific positions of Fc protein chains. In certainembodiments, the modified Fc proteins are made by solid phase synthesis,semi-synthesis, in vivo translation, in vitro translation or cell-freetranslation.

In another aspect, provided herein are methods of making the conjugatesof the modified Fc protein (also referred to as the Fc proteinconjugates). The Fc protein conjugates can be made by any techniqueapparent to those of skill in the art for incorporating non-naturalamino acids into site-specific positions of Fc protein chains and forlinking the Fc proteins to payload molecules. In certain embodiments,the modified Fc proteins are made by solid phase synthesis,semi-synthesis, in vivo translation, in vitro translation or cell-freetranslation.

In another aspect, provided herein are methods of using the Fc proteinconjugates for therapy. Modified Fc proteins directed to a therapeutictarget can incorporate one or more site-specific non-natural amino acidsaccording to the description herein. These Fc protein conjugates can beused for treating or preventing a disease or condition associated withthe therapeutic target. Advantageously, a site-specific non-naturalamino acid is used to link the Fc protein to a therapeutic payload tofacilitate efficacy. Exemplary Fc protein conjugates, therapeutictargets and diseases or conditions are described herein.

In another aspect, provided herein are methods of using the Fc proteinconjugates for detection. Fc protein conjugates can incorporate one ormore site-specific non-natural amino acids according to the descriptionherein. These modified Fc proteins can be used with a label to signalbinding to the detection target. Advantageously, a site-specificnon-natural amino acid can be used to link the modified Fc protein to alabel to facilitate detection. Exemplary Fc protein conjugates,detection targets and labels are described herein.

In another aspect, provided herein are methods of modifying thestability of payload molecules. Fc proteins can be modified with anon-natural amino acid as described herein to facilitate linking to apayload molecule thereby modifying the stability of the payloadmolecule. For instance, a payload molecule can be linked to an Fcprotein to increase the in vivo stability of the payload molecule.Exemplary payload molecules and linking moieties are described herein.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 provides the structure of exemplary cytotoxic reagent DBCO-MMAF.

FIG. 2 provides a single peak hydrophobic interaction chromatography(HIC) assay profile.

FIG. 3 provides a not-well-resolved (NWR) HIC assay profile.

FIG. 4 provides a well-resolved (WR) HIC assay profile.

FIGS. 5A and 5B depict exemplary HIC traces of two variants (HC T110 andHC S112), showing peaks corresponding to unconjugated, partiallyconjugated and fully conjugated IgGs.

FIGS. 6A-6E depict suppression efficiency and soluble yield comparisondata of antibodies comprising different unnatural amino acids.

FIGS. 7A and 7B depict suppression efficiency and soluble yieldcomparison data of antibodies comprising different unnatural amino acids

FIG. 8 depicts cell killing of CD-30 positive cell lines by exemplarybrentuximab antibody-drug conjugates.

FIG. 9 provides experimental results demonstrating that exemplarybrentuximab antibody-drug conjugates do not kill cells that do notexpress CD-30.

FIG. 10 depicts binding to CD-30 positive cell lines by exemplarybrentuximab (ADCETRIS®) antibody-drug conjugates.

FIGS. 11A, 11B, 11C, and 11D provide the structure of exemplarycytotoxic reagents DBCO-MMAF 2, DBCO-DM4, DBCO-DM4 2, and DBCO-MMAE,respectively.

FIG. 12 provides a graphical representation of the pharmacokinetics ofan exemplary scFv-Fc site-specific antibody-drug conjugate.

FIGS. 13A and 13B depict a graphical representation of the in vivoeffectiveness of exemplary site-specific antibody-drug conjugates toretard tumor growth and/or to regress tumor size in an animal model.

DESCRIPTION OF EXEMPLARY EMBODIMENTS

Provided herein are modified Fc proteins having non-natural amino acidsat one or more site-specific positions, compositions comprising themodified Fc proteins and conjugates thereof, methods of making themodified Fc proteins and conjugates thereof, and methods of their use.

Definitions

When referring to the modified Fc proteins or conjugates thereofprovided herein, the terms used have the following meanings unlessindicated otherwise. Unless defined otherwise, all technical andscientific terms used herein have the same meaning as is commonlyunderstood by one of ordinary skill in the art. In the event that thereis a plurality of definitions for a term herein, those in this sectionprevail unless stated otherwise.

As used herein, the singular forms “a,” “an,” and “the” include theplural reference unless the context clearly indicates otherwise. Thus,for example, reference to an “Fc protein” is a reference to one or moresuch Fc proteins, etc.

The term “substantially pure” with respect to a composition comprising amodified Fc protein refers to a composition that includes at least 80,85, 90 or 95% by weight or, in certain embodiments, 95, 98, 99 or 100%by weight, e.g. dry weight, of the modified Fc protein relative to theremaining portion of the composition. The weight percentage can berelative to the total weight of protein in the composition or relativeto the total weight of Fc proteins in the composition. Purity can bedetermined by techniques apparent to those of skill in the art, forinstance SDS-PAGE.

The term “isolated” refers to an Fc protein a modified Fc protein or aconjugate thereof that is substantially or essentially free ofcomponents that normally accompany or interact with the antibody asfound in its naturally occurring environment or in its productionenvironment, or both. Isolated antibody preparations have less thanabout 30%, less than about 25%, less than about 20%, less than about15%, less than about 10%, less than about 5%, less than about 4%, lessthan about 3%, less than about 2% or less than about 1% of contaminatingprotein by weight, e.g. dry weight.

The term “antibody” refers to any macromolecule that would be recognizedas an antibody by those of skill in the art. Antibodies share commonproperties including binding and at least one polypeptide chain that issubstantially identical to a polypeptide chain that can be encoded byany of the immunoglobulin genes recognized by those of skill in the art.The immunoglobulin genes include, but are not limited to, the κ, λ, α, γ(IgG1, IgG2, IgG3, and IgG4), δ, ε and μ constant region genes, as wellas the immunoglobulin variable region genes. The term includesfull-length antibodies and antibody fragments recognized by those ofskill in the art, and variants thereof. The term further includesglycosylated and aglycosylated antibodies.

The term “antibody fragment” refers to any form of an antibody otherthan the full-length form. Antibody fragments herein include antibodiesthat are smaller components that exist within full-length antibodies,and antibodies that have been engineered. Antibody fragments include butare not limited to Fv, Fc, Fab, and (Fab′)₂, single chain Fv (scFv),diabodies, triabodies, tetrabodies, bifunctional hybrid antibodies,CDR1, CDR2, CDR3, combinations of CDR's, variable regions, frameworkregions, constant regions, and the like (Maynard & Georgiou, 2000, Annu.Rev. Biomed. Eng. 2:339-76; Hudson, 1998, Curr. Opin. Biotechnol.9:395-402).

The term “immunoglobulin (Ig)” refers to a protein consisting of one ormore polypeptides substantially encoded by one of the immunoglobulingenes, or a protein substantially identical thereto in amino acidsequence. Immunoglobulins include but are not limited to antibodies.Immunoglobulins may have a number of structural forms, including but notlimited to full-length antibodies, antibody fragments, and individualimmunoglobulin domains including but not limited to V_(H), Cγ1, Cγ2,Cγ3, V_(L), and C_(L).

The term “immunoglobulin (Ig) domain” refers to a protein domainconsisting of a polypeptide substantially encoded by an immunoglobulingene. Ig domains include but are not limited to V_(H), Cγ1, Cγ2, Cγ3,V_(L), and C_(L).

The term “variable region” of an antibody refers to a polypeptide orpolypeptides composed of the V_(H) immunoglobulin domain, the V_(L)immunoglobulin domains, or the V_(H) and V_(L) immunoglobulin domains.Variable region may refer to this or these polypeptides in isolation, asan Fv fragment, as a scFv fragment, as this region in the context of alarger antibody fragment, or as this region in the context of afull-length antibody or an alternative, non-antibody scaffold molecule.

The term “variable” refers to the fact that certain portions of thevariable domains differ extensively in sequence among antibodies and areresponsible for the binding specificity of each particular antibody forits particular antigen. However, the variability is not evenlydistributed through the variable domains of antibodies. It isconcentrated in three segments called Complementarity DeterminingRegions (CDRs) both in the light chain and the heavy chain variabledomains. The more highly conserved portions of the variable domains arecalled the framework regions (FR). The variable domains of native heavyand light chains each comprise four FR regions, largely adopting aβ-sheet configuration, connected by three or four CDRs, which form loopsconnecting, and in some cases forming part of, the β-sheet structure.The CDRs in each chain are held together in close proximity by the FRregions and, with the CDRs from the other chain, contribute to theformation of the antigen binding site of antibodies (see Kabat et al.,Sequences of Proteins of Immunological Interest, 5th Ed. Public HealthService, National Institutes of Health, Bethesda, Md. (1991)).

The constant domains are not typically involved directly in binding anantibody to an antigen, but exhibit various effector functions.Depending on the amino acid sequence of the constant region of theirheavy chains, antibodies or immunoglobulins can be assigned to differentclasses. There are five major classes of immunoglobulins: IgA, IgD, IgE,IgG and IgM, and several of these may be further divided into subclasses(isotypes), e.g. IgG1, IgG2, IgG3, and IgG4; IgA1 and IgA2. The heavychain constant regions that correspond to the different classes ofimmunoglobulins are called α, δ, ε, γ and μ, respectively. Of thevarious human immunoglobulin classes, only human IgG1, IgG2, IgG3 andIgM are known to activate complement.

The term “Fc protein” refers to any macromolecule that would berecognized as an Fc protein by those of skill in the art. An Fc proteingenerally corresponds to the Fc region (fragment crystallizable region)of an antibody, as known to those of skill in the art. Fc proteins sharecommon properties including binding to one or more Fc receptors. Fcproteins corresponding to IgG, IgG and IgD antibodies comprise domainscorresponding to the C_(H)2 and C_(H)3 domains of these antibodies. Fcproteins corresponding to IgM and IgE antibodies comprise domainscorresponding to C_(H)2, C_(H)3 and C_(H)4 domains of these antibodies.In certain embodiments, the Fc proteins are glycosylated. In certainembodiments, the Fc proteins are dimers. In certain embodiments, the Fcproteins are homodimers. In certain embodiments, the Fc proteins areheterodimers. In certain embodiments, the dimers are linked via adisulfide bond. In certain embodiments, the dimers are linked by anamino acid or a peptide bridge. In certain embodiments, Fc proteins donot comprise a variable domain. In certain embodiments, Fc proteins donot comprise a light chain. In certain embodiments, Fc proteins do notcomprise a variable domain or a light chain.

The term “conjugate” refers to any moiety that can be connected to amodified Fc protein. In some embodiments, the terms “conjugate” and“payload” are used interchangeably. A conjugate can be a small moleculeor a macromolecule. In some embodiments, the conjugate is a bioactivemolecule including but not limited to a protein, a peptide, a nucleicactive or a hybrid thereof. In some embodiments, the conjugate is apolymer such as polyethylene glycol. In some embodiments, a conjugate isa therapeutic agent, including a commercially available drug. In someembodiments, a conjugate is a label that can recognize and bind tospecific targets, such as a molecular payload that is harmful to targetcells or a label useful for detection or diagnosis. In some embodiments,the conjugate is connected to an Fc protein via a linker. In someembodiments, the conjugate is directly connected to an Fc proteinwithout a linker.

The term “variant protein sequence” refers to a protein sequence thathas one or more residues that differ in amino acid identity from anothersimilar protein sequence. Said similar protein sequence may be thenatural wild type protein sequence, or another variant of the wild typesequence. Variants include proteins that have one or more amino acidinsertions, deletions or substitutions. Variants also include proteinsthat have one or more post-translationally modified amino acids.

The term “parent antibody” refers to an antibody known to those of skillin the art that is modified according to the description providedherein. The modification can be physical, i.e., chemically orbiochemically replacing or modifying one or more amino acids of theparent antibody to yield an antibody within the scope of the presentdescription. The modification can also be conceptual, i.e., using thesequence of one or more polypeptide chains of the parent antibody todesign an antibody comprising one or more site-specific non-naturalamino acids according to the present description. Parent antibodies canbe naturally occurring antibodies or antibodies designed or developed ina laboratory. Parent antibodies can also be artificial or engineeredantibodies, e.g., chimeric or humanized antibodies.

The term “conservatively modified variant” refers to an Fc protein thatdiffers from a related Fc protein by conservative substitutions in aminoacid sequence. One of skill will recognize that individualsubstitutions, deletions or additions to a peptide, polypeptide, orprotein sequence which alters, adds or deletes a single amino acid or asmall percentage of amino acids in the encoded sequence is a“conservatively modified variant” where the alteration results in thesubstitution of an amino acid with a chemically similar amino acid.Conservative substitution tables providing functionally similar aminoacids are well known in the art. Such conservatively modified variantsare in addition to and do not exclude polymorphic variants, interspecieshomologs, and alleles of the invention.

The following eight groups each contain amino acids that areconservative substitutions for one another:

1) Alanine (A), Glycine (G);

2) Aspartic acid (D), Glutamic acid (E);

3) Asparagine (N), Glutamine (Q);

4) Arginine (R), Lysine (K), Histidine (H);

5) Isoleucine (I), Leucine (L), Methionine (M), Valine (V);

6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W);

7) Serine (S), Threonine (T); and

8) Cysteine (C), Methionine (M)

(see, e.g., Creighton, Proteins: Structures and Molecular Properties (WH Freeman & Co.; 2nd edition (December 1993)

The terms “identical” or “identity,” in the context of two or morepolypeptide sequences, refer to two or more sequences or subsequencesthat are the same. Sequences are “substantially identical” if they havea percentage of amino acid residues or nucleotides that are the same(i.e., about 60% identity, optionally about 65%, about 70%, about 75%,about 80%, about 85%, about 90%, or about 95% identity over a specifiedregion), when compared and aligned for maximum correspondence over acomparison window, or designated region as measured using one of thefollowing sequence comparison algorithms or by manual alignment andvisual inspection. The identity can exist over a region that is at leastabout 50 amino acids or nucleotides in length, or over a region that is75-100 amino acids or nucleotides in length, or, where not specified,across the entire sequence or a polypeptide. In the case of antibodies,identity can be measured outside the variable CDRs. Optimal alignment ofsequences for comparison can be conducted, including but not limited to,by the local homology algorithm of Smith and Waterman (1970) Adv. Appl.Math. 2:482c, by the homology alignment algorithm of Needleman andWunsch (1970) J. Mol. Biol. 48:443, by the search for similarity methodof Pearson and Lipman (1988) Proc. Nat'l. Acad. Sci. USA 85:2444, bycomputerized implementations of these algorithms (GAP, BESTFIT, FASTA,and TFASTA in the Wisconsin Genetics Software Package, Genetics ComputerGroup, 575 Science Dr., Madison, Wis.); or by manual alignment andvisual inspection (see, e.g., Ausubel et al., Current Protocols inMolecular Biology (1995 supplement)).

Examples of algorithms that are suitable for determining percentsequence identity and sequence similarity include the BLAST and BLAST2.0 algorithms, which are described in Altschul et al. (1977) Nuc. AcidsRes. 25:3389-3402, and Altschul et al. (1990) J. Mol. Biol. 215:403-410,respectively. Software for performing BLAST analyses is publiclyavailable through the National Center for Biotechnology Information. TheBLAST algorithm parameters W, T, and X determine the sensitivity andspeed of the alignment. The BLASTN program (for nucleotide sequences)uses as defaults a wordlength (W) of 11, an expectation (E) or 10, M=5,N=−4 and a comparison of both strands. For amino acid sequences, theBLASTP program uses as defaults a wordlength of 3, and expectation (E)of 10, and the BLOSUM62 scoring matrix (see Henikoff and Henikoff (1989)Proc. Natl. Acad. Sci. USA 89:10915) alignments (B) of 50, expectation(E) of 10, M=5, N=−4, and a comparison of both strands. The BLASTalgorithm is typically performed with the “low complexity” filter turnedoff. In some embodiments, the BLAST algorithm is typically performedwith the “low complexity” filter turned on.

The BLAST algorithm also performs a statistical analysis of thesimilarity between two sequences (see, e.g., Karlin and Altschul (1993)Proc. Natl. Acad. Sci. USA 90:5873-5787). One measure of similarityprovided by the BLAST algorithm is the smallest sum probability (P(N)),which provides an indication of the probability by which a match betweentwo nucleotide or amino acid sequences would occur by chance. Forexample, a nucleic acid is considered similar to a reference sequence ifthe smallest sum probability in a comparison of the test nucleic acid tothe reference nucleic acid is less than about 0.2, more preferably lessthan about 0.01, and most preferably less than about 0.001.

The term “amino acid” refers to naturally occurring and non-naturallyoccurring amino acids, as well as amino acids such as proline, aminoacid analogs and amino acid mimetics that function in a manner similarto the naturally occurring amino acids.

Naturally encoded amino acids are the proteinogenic amino acids known tothose of skill in the art. They include the 20 common amino acids(alanine, arginine, asparagine, aspartic acid, cysteine, glutamine,glutamic acid, glycine, histidine, isoleucine, leucine, lysine,methionine, phenylalanine, proline, serine, threonine, tryptophan,tyrosine, and valine) and the less common pyrrolysine andselenocysteine. Naturally encoded amino acids include post-translationalvariants of the 22 naturally occurring amino acids such as prenylatedamino acids, isoprenylated amino acids, myrisoylated amino acids,palmitoylated amino acids, N-linked glycosylated amino acids, O-linkedglycosylated amino acids, phosphorylated amino acids and acylated aminoacids.

The term “non-natural amino acid” refers to an amino acid that is not aproteinogenic amino acid, or a post-translationally modified variantthereof. In particular, the term refers to an amino acid that is not oneof the 20 common amino acids or pyrrolysine or selenocysteine, orpost-translationally modified variants thereof.

A “functional Releasing Factor 1 (RF1) protein” refers to RF1 thatretains activity equal to or substantially similar to wild-type orunmodified RF1 protein. Functional RF1 activity can be tested, forexample, by measuring the growth rate of bacteria expressing themodified RF1 protein, and comparing the growth rate to bacteriaexpressing wild-type or unmodified RF1. Functional RF1 activity can alsobe tested, for example, by the ability of the modified RF1 protein toreduce orthogonal tRNA incorporation of a nnAA at a specified positionin an mRNA encoding a target protein, thereby increasing the amount ofpremature chain termination (i.e., increasing the amount of truncatedprotein).

An “attenuated Releasing Factor 1 (RF1) protein” refers to a modifiedRF1 that retains reduced activity relative to wild-type or unmodifiedRF1 protein. RF1 activity can be tested, for example, by measuring thegrowth rate of bacteria expressing the modified RF1 protein, andcomparing the growth rate to bacteria expressing wild-type or unmodifiedRF1. RF1 activity can also be tested, for example, by the ability of themodified RF1 protein to reduce orthogonal tRNA incorporation of a nnAAat a specified position in an mRNA encoding a target protein, therebyincreasing the amount of premature chain termination (i.e., increasingthe amount of truncated protein). In some embodiments, the attenuatedRF1 protein comprises transcriptional modifications; for example, theexpression level of the RF1 protein (wild type or modified) can bereduced to achieve attenuation. The reduction can also achieved by usingRNAi technologies. In some embodiments, the attenuated RF1 proteincomprises translational modifications; for example, the amount of thesynthesized RF1 protein (wild type or modified) can be reduced toachieve attenuation, e.g., by increasing the rate at which the proteinis digested by protease via insertion of protease-specific sequence intothe RF1 sequence.

Fc Proteins

Provided herein are modified Fc proteins comprising one or morenon-natural amino acid residues at site-specific positions in the aminoacid sequence of at least one polypeptide chain.

The modified Fc protein can share high sequence identity with any Fcprotein recognized by those of skill in the art, i.e. a parent Fcprotein. In some embodiments, a parent Fc protein is an Fc fragment froman immunoglobulin that can be isolated from a subject. In certainembodiments, the amino acid sequence of the Fc protein is identical tothe amino acid sequence of the parent Fc protein, other than thenon-natural amino acids at site-specific position. In furtherembodiments, the modified Fc protein provided herein can have one ormore insertions, deletions or mutations relative to the parent Fcprotein in addition to the one or more non-natural amino acids at thesite-specific positions. In certain embodiments, the modified Fc proteinprovided herein can have a unique primary sequence, so long as it wouldbe recognized as an Fc protein by those of skill in the art.

In certain embodiments, the Fc protein comprising one or morenon-natural amino acid residues at site-specific positions has highsequence identity to any parent Fc protein described herein or known tothose of skill in the art. In certain embodiments, the Fc protein issubstantially identical to a parent Fc protein described herein. Incertain embodiments, the Fc protein has about 60% identity, about 65%,about 70%, about 75%, about 80%, about 85%, about 90%, or about 95%identity to a parent Fc protein. In certain embodiments, the Fc proteinhas greater than about 65%, greater than about 70%, greater than about75%, greater than about 80%, greater than about 85%, greater than about90%, or greater than about 95% identity to a parent Fc protein. Fcproteins are substantially identical if at least one polypeptide chainof Fc protein has sequence identity to a corresponding Fc protein chainof another Fc protein. In certain embodiments, the Fc protein comprisingone or more non-natural amino acid residues at site-specific positionshas at least one polypeptide chain having greater than about 65%,greater than about 70%, greater than about 75%, greater than about 80%,greater than about 85%, greater than about 90%, or greater than about95% identity to any of SEQ ID NO:1 and 4-6 over at least one domain, atleast two domains or at least three domains. In certain embodiments,percent identity is over at least one chain. In certain embodiments,percent identity is over at least two chains.

In some embodiments and similar to a parent Fc protein, the modified Fcprotein can be a monomer or a dimer. In certain embodiments, themodified Fc protein is a dimer comprising polypeptides corresponding toone or more constant domains of an antibody. In some embodiments, Fcproteins corresponding to IgG, IgG and IgD antibodies comprise domainscorresponding to the C_(H)2 and C_(H)3 domains of these antibodies. Insome embodiments, Fc proteins corresponding to IgM and IgE antibodiescomprise domains corresponding to C_(H)2, C_(H)3 and C_(H)4 domains ofthese antibodies. Each polypeptide chain can be linked to the otherpolypeptide chain by one or more covalent disulfide bonds. Eachpolypeptide chain can also have one or more intrachain disulfide bonds.In certain embodiments, the Fc proteins are glycosylated. In someembodiments, the linker sequence includes a single amino acid. In otherembodiments, the linker sequence includes a peptide. In someembodiments, linker peptide sequences can be random linker sequencesthat offer structural flexibilities. In some embodiments, linker peptidesequences can be selected based on the structures of the individualpeptide chains, by, for example, searching libraries of protein orpeptide structures. In certain embodiments, a linker peptide sequence isselected to best join the structures of individual peptide chains of thedimer or multimer. In some embodiments, non-natural amino acids can beincorporated into the linker sequence. In some embodiments, the linkercan include non-amino acids such as alkanes, lipid or fat molecules.

As is known to those of skill in the art, Fc proteins typically havebinding affinity for Fc receptors in vivo.

The modified Fc proteins provided herein can have sequences that aresimilar or identical to those of any Fc protein form known to those ofskill in the art. They can be full-length, or fragments. Exemplary fulllength Fc proteins correspond to the Fc domains of IgA, IgA1, IgA2, IgD,IgE, IgG, IgG1, IgG2, IgG3, IgG4 or IgM.

The modified Fc proteins provided herein comprise at least onenon-natural amino acid. The non-natural amino acid can be anynon-natural amino acid known to those of skill in the art. Exemplarynon-natural amino acids are described in the sections below.

The non-natural amino acids are positioned at selected locations in apolypeptide chain of the parent Fc protein. These locations wereidentified as providing optimum sites for substitution with thenon-natural amino acids. Each site is capable of bearing a non-naturalamino acid with optimum structure, function and/or methods for producingthe modified Fc protein.

In certain embodiments, a site-specific position for substitutionprovides a modified Fc protein that is more stable compared to an Fcprotein without the site-specific non-natural amino acid. Stability canbe measured by any technique apparent to those of skill in the art.

In certain embodiments, a site-specific position for substitutionprovides a modified Fc protein that is has optimal functional propertiescompared to an Fc protein without the site-specific non-natural aminoacid. For instance, the modified Fc protein can show little or no lossof binding affinity for Fc receptor compared to an Fc protein withoutthe site-specific non-natural amino acid. In certain embodiments, themodified Fc protein can show enhanced binding compared to an Fc proteinwithout the site-specific non-natural amino acid.

In certain embodiments, a site-specific position for substitutionprovides a modified Fc protein that can be made advantageously. Forinstance, in certain embodiments, the modified Fc protein showsadvantageous properties in its methods of synthesis compared to an Fcprotein without the site-specific non-natural amino acid, discussedbelow. In certain embodiments, the modified Fc protein can show littleor no loss in yield in production compared to an Fc protein without thesite-specific non-natural amino acid. In certain embodiments, themodified Fc protein can show enhanced yield in production compared to anFc protein without the site-specific non-natural amino acid. In certainembodiments, the modified Fc protein can show little or no loss of tRNAsuppression, described below, compared to an Fc protein without thesite-specific non-natural amino acid. In certain embodiments, themodified Fc protein can show enhanced tRNA suppression, described below,in production compared to an Fc protein without the site-specificnon-natural amino acid.

In certain embodiments, a site-specific position for substitutionprovides a modified Fc protein that has advantageous solubility comparedto an Fc protein without the site-specific non-natural amino acid. Incertain embodiments, the modified Fc protein can show little or no lossin solubility compared to an Fc protein without the site-specificnon-natural amino acid. In certain embodiments, the modified Fc proteincan show enhanced solubility compared to an Fc protein without thesite-specific non-natural amino acid.

In certain embodiments, a site-specific position for substitutionprovides a modified Fc protein that has advantageous expression comparedto an Fc protein without the site-specific non-natural amino acid. Incertain embodiments, the modified Fc protein can show little or no lossin expression compared to an Fc protein without the site-specificnon-natural amino acid. In certain embodiments, the modified Fc proteincan show enhanced expression compared to an Fc protein without thesite-specific non-natural amino acid.

In certain embodiments, a site-specific position for substitutionprovides a modified Fc protein that has advantageous folding compared toan Fc protein without the site-specific non-natural amino acid. Incertain embodiments, the modified Fc protein can show little or no lossin proper folding compared to an Fc protein without the site-specificnon-natural amino acid. In certain embodiments, the modified Fc proteincan show enhanced folding compared to an Fc protein without thesite-specific non-natural amino acid.

In certain embodiments, a site-specific position for substitutionprovides a modified Fc protein that is capable of advantageousconjugation compared to an Fc protein without the site-specificnon-natural amino acid. As described below, several non-natural aminoacids have side chains or functional groups that facilitate conjugationof the modified Fc protein to a second agent, either directly or via alinker. In certain embodiments, the modified Fc protein can showenhanced conjugation efficiency compared to an Fc protein without thesame or other non-natural amino acids at other positions. In certainembodiments, the modified Fc protein can show enhanced conjugation yieldcompared to an Fc protein without the same or other non-natural aminoacids at other positions. In certain embodiments, the modified Fcprotein can show enhanced conjugation specificity compared to an Fcprotein without the same or other non-natural amino acids at otherpositions.

The one or more non-natural amino acids are located at selectedsite-specific positions in at least one polypeptide chain of themodified Fc protein. If the modified Fc protein is a dimer or multimer,at least one non-natural amino acid can be in either polypeptide of thedimer. Further, at least one non-natural amino acid can be in any domainof a polypeptide of an Fc protein. Of course, a modified Fc protein cancomprise a plurality of non-natural amino acids at site-specificpositions, in any domain and/or in any polypeptide.

In certain embodiments, the modified Fc proteins provided hereincomprise one non-natural amino acid at a site-specific position. Incertain embodiments, the modified Fc proteins provided herein comprisetwo non-natural amino acids at site-specific positions. In certainembodiments, the modified Fc proteins provided herein comprise threenon-natural amino acids at site-specific positions. In certainembodiments, the modified Fc proteins provided herein comprise more thanthree non-natural amino acids at site-specific positions.

The site-specific positions for substituting can be described accordingto any Fc protein nomenclature system known to those of skill in theart. In the EU numbering system, these positions are at heavy chainresidues H238, H239, H241, H243, H246, H262, H264, H265, H267, H268,H269, H270, H271, H272, H274, H275, H278, H280, H281, H282, H283, H286,H289, H292, H293, H294, H295, H296, H297, H298, H299, H300, H301, H303,H305, H317, H320, H324, H326, H327, H329, H330, H332, H333, H334, H335,H337, H339, H340, H342, H344, H355, H356, H358, H359, H360, H375, H383,H384, H386, H389, H392, H398, H404, H420, H421, H436, and H438. In otherwords, provided herein are modified Fc proteins comprising one or morenon-natural amino acids at one or more positions selected from EUresidues H238, H239, H241, H243, H246, H262, H264, H265, H267, H268,H269, H270, H271, H272, H274, H275, H278, H280, H281, H282, H283, H286,H289, H292, H293, H294, H295, H296, H297, H298, H299, H300, H301, H303,H305, H317, H320, H324, H326, H327, H329, H330, H332, H333, H334, H335,H337, H339, H340, H342, H344, H355, H356, H358, H359, H360, H375, H383,H384, H386, H389, H392, H398, H404, H420, H421, H436, and H438.

In certain embodiments, provided herein are modified Fc proteinscomprising one or more non-natural amino acids at one or more positionsselected from EU residues H239, H241, H246, H267, H268, H269, H270,H271, H272, H274, H275, H280, H281, H282, H283, H286, H289, H292, H293,H294, H295, H296, H297, H298, H299, H300, H301, H303, H305, H317, H320,H324, H326, H327, H329, H330, H332, H333, H334, H335, H337, H339, H340,H342, H344, H355, H359, H375, H386, H389, H392, H398, H404, H420, H421,and H438.

In certain embodiments, provided herein are modified Fc proteinscomprising one or more non-natural amino acids at one or more positionsselected from EU residues H239, H293, H334, H355, H359, and H389.

In certain embodiments, provided herein are modified Fc proteinscomprising one or more non-natural amino acids at one or more positionsselected from EU residues H241, H246, H267, H268, H269, H270, H271,H272, H274, H275, H280, H281, H282, H283, H286, H289, H292, H294, H295,H296, H297, H298, H299, H300, H301, H303, H305, H317, H320, H324, H326,H327, H329, H330, H332, H333, H335, H337, H339, H342, H344, H355, H375,H386, H392, H398, H420, H421, H340, H404, and H438.

In certain embodiments, provided herein are modified Fc proteinscomprising one or more non-natural amino acids at one or more positionsselected from EU residues H238, H243, H262, H264, H265, H278, H342,H356, H358, H360, H383, H384, H404 and H436.

In certain embodiments, provided herein are modified Fc proteinscomprising one or more non-natural amino acids at one or more positionsselected from EU residues corresponding to H292-H301, H303, and H305.

In the EU numbering system, these positions are at heavy chain residuesH238, H239, H241, H243, H246, H262, H264, H265, H267, H268, H269, H270,H271, H272, H274, H275, H278, H280, H281, H282, H283, H286, H289, H292,H293, H294, H295, H296, H297, H298, H299, H300, H301, H303, H305, H317,H320, H324, H326, H327, H329, H330, H332, H333, H334, H335, H337, H339,H340, H342, H344, H355, H356, H358, H359, H360, H375, H383, H384, H386,H389, H392, H398, H404, H420, H421, H436, and H438. In other words,provided herein are modified Fc proteins comprising one or morenon-natural amino acids at one or more positions selected from EUresidues H238, H239, H241, H243, H246, H262, H264, H265, H267, H268,H269, H270, H271, H272, H274, H275, H278, H280, H281, H282, H283, H286,H289, H292, H293, H294, H295, H296, H297, H298, H299, H300, H301, H303,H305, H317, H320, H324, H326, H327, H329, H330, H332, H333, H334, H335,H337, H339, H340, H342, H344, H355, H356, H358, H359, H360, H375, H383,H384, H386, H389, H392, H398, H404, H420, H421, H436, and H438.

In certain embodiments, provided herein are modified Fc proteinscomprising one or more non-natural amino acids at one or more positionsselected from EU residues H239, H241, H246, H267, H268, H269, H270,H271, H272, H274, H275, H280, H281, H282, H283, H286, H289, H292, H293,H294, H295, H296, H297, H298, H299, H300, H301, H303, H305, H317, H320,H324, H326, H327, H329, H330, H332, H333, H334, H335, H337, H339, H340,H342, H344, H355, H359, H375, H386, H389, H392, H398, H404, H420, H421,and H438.

In certain embodiments, provided herein are modified Fc proteinscomprising one or more non-natural amino acids at one or more positionsselected from EU residues H239, H293, H334, H342, H355, H359, and H389.

In certain embodiments, provided herein are modified Fc proteinscomprising one or more non-natural amino acids at one or more positionsselected from EU residues corresponding to H292-H301, H303, and H305.

In certain embodiments, provided herein are modified Fc proteinscomprising one or more non-natural amino acids at one or more positionsselected from EU residues H241, H246, H267, H268, H269, H270, H271,H272, H274, H275, H280, H281, H282, H283, H286, H289, H292, H294, H295,H296, H297, H298, H299, H300, H301, H303, H305, H317, H320, H324, H326,H327, H329, H330, H332, H333, H335, H337, H339, H342, H344, H355, H375,H386, H389, H392, H398, H404, H420, H421, H340 and H438.

In certain embodiments, provided herein are modified Fc proteinscomprising one or more non-natural amino acids at one or more positionsselected from EU residues H238, H243, H262, H264, H265, H278, H342,H356, H358, H360, H383, H384, H389, H404, and H436.

The site-specific positions can also be identified relative to the aminoacid sequences of the polypeptide chains of a reference Fc protein. Forexample, the amino acid sequence of a reference heavy chain is providedin SEQ ID NO:1. In the exemplary reference heavy chain, thesite-specific positions are at residues 241, 242, 244, 246, 249, 265,267, 268, 270, 271, 272, 273, 274, 275, 277, 278, 281, 283, 284, 285,286, 289, 292, 295, 296, 297, 298, 299, 300, 301, 302, 303, 304, 306,308, 320, 323, 327, 329, 330, 332, 333, 335, 336, 337, 338, 340, 342,343, 345, 347, 358, 359, 361, 362, 363, 378, 386, 387, 389, 392, 395,401, 407, 423, 424, 439 and 441. In other words, provided herein are Fcproteins comprising one or more non-natural amino acids at one or morepositions selected from those corresponding to residues 241, 242, 244,246, 249, 265, 267, 268, 270, 271, 272, 273, 274, 275, 277, 278, 281,283, 284, 285, 286, 289, 292, 295, 296, 297, 298, 299, 300, 301, 302,303, 304, 306, 308, 320, 323, 327, 329, 330, 332, 333, 335, 336, 337,338, 340, 342, 343, 345, 347, 358, 359, 361, 362, 363, 378, 386, 387,389, 392, 395, 401, 407, 423, 424, 439 and 441 of the representativeheavy chain polypeptide according to SEQ ID NO:1.

In certain embodiments, provided herein are modified Fc proteinscomprising one or more non-natural amino acids at one or more positionsselected from those corresponding to residues 242, 244, 249, 270, 271,272, 273, 274, 275, 277, 278, 283, 284, 285, 286, 289, 292, 295, 296,297, 298, 299, 300, 301, 302, 303, 304, 306, 308, 320, 323, 327, 329,330, 332, 333, 335, 336, 337, 338, 340, 342, 343, 345, 347, 358, 362,378, 389, 392, 395, 401, 407, 423, 424, and 441 of the representativeheavy chain polypeptide according to SEQ ID NO:1.

In certain embodiments, provided herein are modified Fc proteinscomprising one or more non-natural amino acids at one or more positionsselected from those corresponding to residues 242, 296, 337, 345, 358,362, and 392 of the representative heavy chain polypeptide according toSEQ ID NO:1.

In certain embodiments, provided herein are modified Fc proteinscomprising one or more non-natural amino acids at one or more positionsselected from those corresponding to residues 244, 249, 270, 271, 272,273, 274, 275, 277, 278, 283, 284, 285, 286, 289, 292, 295, 297, 298,299, 300, 301, 302, 303, 304, 306, 308, 320, 323, 327, 329, 330, 332,333, 335, 336, 338, 340, 342, 343, 345, 347, 358, 378, 389, 395, 401,407, 423, 424, and 441 of the representative heavy chain polypeptideaccording to SEQ ID NO:1.

In certain embodiments, provided herein are modified Fc proteinscomprising one or more non-natural amino acids at one or more positionsselected from those corresponding to residues 241, 246, 265, 267, 268,281, 345, 359, 361, 363, 386, 387, 407, and 439 of the representativeheavy chain polypeptide according to SEQ ID NO:1.

In certain embodiments, provided herein are modified Fc proteinscomprising one or more non-natural amino acids at one or more positionsselected from those corresponding to 242, 244, 270, 273, 274, 275, 285,289, 295, 296, 299, 300, 301, 332, 333, 337, 338, 343, 345, 358, 362,389, 407, 423, 424, and 441 of the representative heavy chainpolypeptide according to SEQ ID NO:1.

In certain embodiments, provided herein are modified Fc proteinscomprising one or more non-natural amino acids at one or more positionsselected from those corresponding to residues 292-301, 303, and 305 ofthe representative heavy chain polypeptide according to SEQ ID NO:1.

In certain embodiments, provided herein are modified Fc proteinscomprising one or more non-natural amino acids at one or more positionsselected from those corresponding to 222, 285, 292, 299, 333, 338, 364,403, 407, 425, 443, 263, 270, 271, 275, 277, 295, 296, 300, 301, 306,308, 335, 336, 337, 343, 344, 345, 346, 358, 365, 389, 395, 407, 427,441, 445, and 446 of the representative heavy chain polypeptideaccording to SEQ ID NO:1.

In certain embodiments, provided herein are modified Fc proteinscomprising one or more non-natural amino acids at one or more positionsselected from those corresponding to 222, 285, 299, 338, 364, 425, 443,270, 275, 277, 295, 296, 300, 301, 306, 308, 337, 343, 344, 345, 346,358, 365, 395, 407, 427, 441, 445, and 446 of the representative heavychain polypeptide according to SEQ ID NO:1.

In certain embodiments, provided herein are modified Fc proteinscomprising one or more non-natural amino acids at one or more positionsselected from those corresponding to 222, 285, 299, 364, 425, 443, 270,275, 296, 300, 301, 306, 308, 337, 343, 344, 345, 358, 365, 395, 407,427, 441, 445, and 446 of the representative heavy chain polypeptideaccording to SEQ ID NO:1.

In certain embodiments, provided herein are modified Fc proteinscomprising one or more non-natural amino acids at one or more positionsselected from those corresponding to 222, 285, 299, 425, 443, 270, 275,296, 300, 301, 306, 308, 337, 343, 344, 345, 358, 395, 407, 427, 441,445, and 446 of the representative heavy chain polypeptide according toSEQ ID NO:1.

In certain embodiments, provided herein are modified Fc proteinscomprising one or more non-natural amino acids at one or more positionsselected from those corresponding to 222, 285, 299, 270, 296, 300, 301,306, 308, 337, 343, 344, 395, 407, and 441 of the representative heavychain polypeptide according to SEQ ID NO:1.

In certain embodiments, provided herein are modified Fc proteinscomprising one or more non-natural amino acids at one or more positionsselected from those corresponding to 224, 225, 228, 230, 233, 234, 235and 239 of the representative heavy chain polypeptide according to SEQID NO:1.

In certain embodiments, provided herein are modified Fc proteinscomprising one or more non-natural amino acids at one or more positionsselected from those corresponding to 224, 225, 228, 230, 233, 234, 235and 239 of the representative heavy chain polypeptide according to SEQID NO:1.

In certain embodiments, provided herein are modified Fc proteinscomprising one or more non-natural amino acids at one or more positionsselected from those corresponding to 230, 233, 234, 235, and 239 of therepresentative heavy chain polypeptide according to SEQ ID NO:1.

In certain embodiments, provided herein are modified Fc proteinscomprising one or more non-natural amino acids at one or more positionsselected from those corresponding to 224, and 225 of the representativeheavy chain polypeptide according to SEQ ID NO:1.

In certain embodiments, the modified Fc protein comprises a polypeptidechain that can be described by the following formula (I):Xaa¹- . . . -(Naa^(p(i)))_(n)- . . . -Xaa^(q)  (I).In Formula (I), each Xaa represents an amino acid in the polypeptidechain of any identity. In other words, each Xaa can be any amino acid,typically any naturally occurring amino acid, or a variant thereof. Thesuperscript to the right of each Xaa represents the position of theamino acid within the primary sequence of the polypeptide chain. Xaa¹represents the first, or N-terminal, amino acid in the polypeptidechain, and Xaa^(q) represents the last, or C-terminal, amino acid in thepolypeptide chain. The variable q is an integer that is equal to thetotal number of amino acids in the polypeptide chain. Each Naarepresents a non-natural amino acid within the polypeptide chain. Usefulnon-natural amino acids are described in the sections below. The integern represents the number of non-natural amino acids in the polypeptidechain. In typical embodiments, n is an integer greater than 1. Eachinteger p(i) is greater than 1 and less than q, and the variable i is aninteger that varies from 1 to n. Each integer p(i) represents asite-specific location in the amino acid sequence for the correspondingNaa. Each site specific location p(i) is optimal for substitution of anaturally occurring amino acid with a non-natural amino acid, such asNaa^(p(i)), according to the techniques described herein.

Analysis of the data from TAG screening as presented in the Examplesallowed selection of sites which are ideal candidates for making Fcprotein conjugates on the basis of cell killing, expression levels,drug-to-antibody ratio, solvent accessibility and (where available)thermal stability. The most preferred sites have lower solventaccessibility, and higher thermostability than the other testedvariants. The preferred sites for nnAA incorporation are listed in TableI, below.

TABLE I Preferred Sites for nnAA Incorporation Site of nnAA PreferenceHC-F404 Most Preferred HC-F241 Most Preferred HC-K222 More Preferred

Accordingly, in certain embodiments, provided herein are antibodiescomprising one or more non-natural amino acids at one or more positionsselected from the group consisting of heavy chain or light chainresidues HC-F404, HC-F241 and HC-K222 according to the EU numberingscheme, or a post-translationally modified variant thereof

In certain embodiments, provided herein are antibodies comprising one ormore non-natural amino acids at one or more positions selected from thegroup consisting of heavy chain or light chain residues—according to theEU numbering scheme, or a post-translationally modified variant thereof

In certain embodiments, provided herein are antibodies comprising one ormore non-natural amino acids at one or more positions selected from thegroup consisting of heavy chain or light chain residues—according to theEU numbering scheme, or a post-translationally modified variant thereof.

In some embodiments, modified Fc proteins provided herein can be furtherconservatively modified using methods known in the art. Conservativelymodified variants of an Fc protein include one or more insertions,deletions or substitutions that do not disrupt the structure and/orfunction of the Fc protein when evaluated by one of skill in the art. Incertain embodiments, conservatively modified variants include 20 orfewer amino acid insertions, deletions or substitutions. In certainembodiments, conservatively modified variants include 15 or fewer aminoacid insertions, deletions or substitutions. In certain embodiments,conservatively modified variants include 10 or fewer amino acidinsertions, deletions or substitutions. In certain embodiments,conservatively modified variants include 9 or fewer amino acidinsertions, deletions or substitutions. In certain embodiments,conservatively modified variants include 8 or fewer amino acidinsertions, deletions or substitutions. In certain embodiments,conservatively modified variants include 7 or fewer amino acidinsertions, deletions or substitutions. In certain embodiments,conservatively modified variants include 6 or fewer amino acidinsertions, deletions or substitutions. In certain embodiments,conservatively modified variants include 5 or fewer amino acidinsertions, deletions or substitutions. In certain embodiments,conservatively modified variants include 4 or fewer amino acidinsertions, deletions or substitutions. In certain embodiments,conservatively modified variants include 3 or fewer amino acidinsertions, deletions or substitutions. In certain embodiments,conservatively modified variants include 2 or fewer amino acidinsertions, deletions or substitutions. In certain embodiments,conservatively modified variants include 1 amino acid insertion,deletion or substitution. In particular embodiments the substitutionsare conservative, substituting an amino acid within the same class, asdescribed above.

In certain embodiments, the modified Fc proteins can be further modifiedto modulate structure, stability and/or activity. In such embodiments,the modifications can be conservative or other than conservative. Themodifications need only be suitable to the practitioner carrying out themethods and using the compositions described herein. In certainembodiments, the modifications decrease but do not eliminate antigenbinding affinity. In certain embodiments, the modifications increaseantigen binding affinity. In certain embodiments, the modificationsenhance structure or stability of the Fc protein. In certainembodiments, the modifications reduce but do not eliminate structure orstability of the Fc protein. In certain embodiments, modified variantsinclude 20 or fewer amino acid insertions, deletions or substitutions.In certain embodiments, modified variants include 15 or fewer amino acidinsertions, deletions or substitutions. In certain embodiments, modifiedvariants include 10 or fewer amino acid insertions, deletions orsubstitutions. In certain embodiments, modified variants include 9 orfewer amino acid insertions, deletions or substitutions. In certainembodiments, modified variants include 8 or fewer amino acid insertions,deletions or substitutions. In certain embodiments, modified variantsinclude 7 or fewer amino acid insertions, deletions or substitutions. Incertain embodiments, modified variants include 6 or fewer amino acidinsertions, deletions or substitutions. In certain embodiments, modifiedvariants include 5 or fewer amino acid insertions, deletions orsubstitutions. In certain embodiments, modified variants include 4 orfewer amino acid insertions, deletions or substitutions. In certainembodiments, modified variants include 3 or fewer amino acid insertions,deletions or substitutions. In certain embodiments, modified variantsinclude 2 or fewer amino acid insertions, deletions or substitutions. Incertain embodiments, modified variants include 1 amino acid insertion,deletion or substitution.

Also within the scope are post-translationally modified variants ofmodified Fc proteins. Any of the modified Fc proteins provided hereincan be post-translationally modified in any manner recognized by thoseof skill in the art. Typical post-translational modifications for Fcproteins include interchain disulfide bonding, intrachain disulfidebonding, N-linked glycosylation and proteolysis. Also provided hereinare other post-translationally modified Fc proteins having modificationssuch as phosphorylation, O-linked glycosylation, methylation,acetylation, lipidation, GPI anchoring, myristoylation and prenylation.The post-translational modification can occur during production, invivo, in vitro or otherwise. In certain embodiments, thepost-translational modification can be an intentional modification by apractitioner, for instance, using the methods provided herein.

Further included within the scope are modified Fc proteins fused tofurther peptides or polypeptides. Exemplary fusions include, but are notlimited to, e.g., a methionyl Fc protein in which a methionine is linkedto the N-terminus of the Fc protein resulting from the recombinantexpression, fusions for the purpose of purification (including but notlimited to, to poly-histidine or affinity epitopes), fusions for thepurpose of linking to other biologically active molecules, fusions withserum albumin binding peptides, and fusions with serum proteins such asserum albumin. The modified Fc proteins may comprise protease cleavagesequences, reactive groups, Fc protein-binding domains (including butnot limited to, FLAG or poly-His) or other affinity based sequences(including but not limited to, FLAG, poly-His, GST, etc.). The modifiedFc proteins may also comprise linked molecules (including but notlimited to, biotin) that improve detection (including but not limitedto, GFP), purification or other features of the modified Fc protein. Incertain embodiments, the modified Fc proteins comprise a C-terminalaffinity sequence that facilitates purification of full length Fcproteins. In certain embodiments, such C-terminal affinity sequence is apoly-His sequence, e.g., a 6-His sequence.

The modified Fc protein can have any Fc protein form recognized by thoseof skill in the art. The Fc protein can comprise a single polypeptidechain. The modified Fc protein can also be in the form of a multimerthat will be recognized by those of skill in the art includinghomodimers, heterodimers, homomultimers, and heteromultimers. Thesemultimers can be linked or unlinked. Useful linkages include interchaindisulfide bonds typical for modified Fc domains. The multimers can alsobe linked by other amino acids, including the non-natural amino acidsintroduced according to the present description. The modified Fc proteincan be based on a parent immunoglobulin such as of any class or subclassincluding IgA, IgA1, IgA2, IgD, IgE, IgG, IgG1, IgG2, IgG3, IgG4 andIgM. In some embodiments, individual peptide chains of a modified Fcprotein dimer or multimer can be joined by a linker sequence to form asingle peptide chain. In some embodiments, the linker sequence includesa single amino acid. In other embodiments, the linker sequence includesa peptide. In some embodiments, linker peptide sequences can be randomlinker sequences that offer structure flexibilities. In someembodiments, linker peptide sequences can be selected based on thestructures of the individual peptide chains, by, for example, searchinglibraries of protein or peptide structures. A linker peptide sequence isselected to best join the structures of individual peptide chains of thedimer or multimer. In some embodiments, non-natural amino acids can beincorporated into the linker sequence as well. In some embodiments, thelinker can include non-amino acids such as alkanes, lipid or fatmolecules. In some embodiments, a single peptide chain bearing thelinker sequence can be synthesized using synthetic methods. In someembodiments, a nucleotide sequence encoding the single peptide chain canbe expressed in a heterologous expression system to produce the desireddimer or multimer.

The modified Fc protein may further be glycosylated or aglycosyated.Accordingly, in certain embodiments, the modified Fc protein isglycosylated. In certain embodiments, the modified Fc protein isaglycosylated. Agylcosylated Fc proteins comprising a non-natural aminoacid may be made, for example, as described in the Examples providedherein or through bacterial expression systems known to those skilled inthe art. Glycosylated Fc proteins comprising a non-natural amino acidmay be made, for example, as described by Axup et al., 2012, Proc. Nat.Acad. Sci. USA 109(40):16101-16106.

Also provided herein are modified Fc proteins that are conjugated to oneor more conjugation moieties. The conjugation moiety can be anyconjugation moiety deemed useful to one of skill in the art. Forinstance, the conjugation moiety can be a polymer, such as polyethyleneglycol, that can improve the stability of the modified Fc protein invitro or in vivo. The conjugation moiety can have therapeutic activity,thereby yielding an Fc protein-drug conjugate. The conjugation moietycan be a molecular payload that is harmful to target cells. Theconjugation moiety can be a label useful for detection or diagnosis. Incertain embodiments, the conjugation moiety is linked to the modified Fcprotein via a direct covalent bond. In certain embodiments, theconjugation moiety is linked to the modified Fc protein via a linker. Inadvantageous embodiments, the conjugation moiety or the linker isattached via one of the non-natural amino acids of the modified Fcprotein. Exemplary conjugation moieties and linkers are discussed in thesections below.

Non-natural Amino Acids

The non-natural amino acid can be any non-natural amino acid known tothose of skill in the art. In some embodiments, the non-naturallyencoded amino acid comprises a functional group. The functional groupcan be any functional group known to those of skill in the art. Incertain embodiments the functional group is a label, a polar group, anon-polar group or a reactive group.

Reactive groups are particularly advantageous for linking furtherfunctional groups to the Fc protein at the site-specific position of theFc protein chain. In certain embodiments, the reactive group is selectedfrom the group consisting of amino, carboxy, acetyl, hydrazino,hydrazido, semicarbazido, sulfanyl, azido and alkynyl.

In certain embodiments, the amino acid residue is according to any ofthe following formulas:

Those of skill in the art will recognize that Fc proteins are generallycomprised of L-amino acids. However, with non-natural amino acids, thepresent methods and compositions provide the practitioner with theability to use L-, D- or racemic non-natural amino acids at thesite-specific positions. In certain embodiments, the non-natural aminoacids described herein include D-versions of the natural amino acids andracemic versions of the natural amino acids.

In the above formulas, the wavy lines indicate bonds that connect to theremainder of the polypeptide chains of the Fc proteins. Thesenon-natural amino acids can be incorporated into polypeptide chains justas natural amino acids are incorporated into the same polypeptidechains. In certain embodiments, the non-natural amino acids areincorporated into the polypeptide chain via amide bonds as indicated inthe formulas.

In the above formulas R designates any functional group withoutlimitation, so long as the amino acid residue is not identical to anatural amino acid residue. In certain embodiments, R can be ahydrophobic group, a hydrophilic group, a polar group, an acidic group,a basic group, a chelating group, a reactive group, a therapeutic moietyor a labeling moiety. In certain embodiments, R is selected from thegroup consisting of R¹NR²R³, R¹C(═O)R², R¹C(═O)OR², R¹N₃, R¹C(≡CH). Inthese embodiments, R¹ is selected from the group consisting of a bond,alkylene, heteroalkylene, arylene, heteroarylene. R² and R³ are eachindependently selected from the group consisting of hydrogen, alkyl andheteroalkyl.

In some embodiments, the non-naturally encoded amino acids include sidechain functional groups that react efficiently and selectively withfunctional groups not found in the common amino acids (including but notlimited to, azido, ketone, aldehyde and aminooxy groups) to form stableconjugates. For example, antigen-binding polypeptide that includes anon-naturally encoded amino acid containing an azido functional groupcan be reacted with a polymer (including but not limited to,poly(ethylene glycol) or, alternatively, a second polypeptide containingan alkyne moiety to form a stable conjugate resulting for the selectivereaction of the azide and the alkyne functional groups to form a Huisgen[3+2] cycloaddition product.

Exemplary non-naturally encoded amino acids that may be suitable for usein the present invention and that are useful for reactions with watersoluble polymers include, but are not limited to, those with carbonyl,aminooxy, hydrazine, hydrazide, semicarbazide, azide and alkyne reactivegroups. In some embodiments, non-naturally encoded amino acids comprisea saccharide moiety. Examples of such amino acids includeN-acetyl-L-glucosaminyl-L-serine, N-acetyl-L-galactosaminyl-L-serine,N-acetyl-L-glucosaminyl-L-threonine,N-acetyl-L-glucosaminyl-L-asparagine and O-mannosaminyl-L-serine.Examples of such amino acids also include examples where thenaturally-occurring N or O-linkage between the amino acid and thesaccharide is replaced by a covalent linkage not commonly found innature—including but not limited to, an alkene, an oxime, a thioether,an amide and the like. Examples of such amino acids also includesaccharides that are not commonly found in naturally-occurring proteinssuch as 2-deoxy-glucose, 2-deoxygalactose and the like.

Many of the non-naturally encoded amino acids provided herein arecommercially available, e.g., from Sigma-Aldrich (St. Louis, Mo., USA),Novabiochem (a division of EMD Biosciences, Darmstadt, Germany), orPeptech (Burlington, Mass., USA). Those that are not commerciallyavailable are optionally synthesized as provided herein or usingstandard methods known to those of skill in the art. For organicsynthesis techniques, see, e.g., Organic Chemistry by Fessendon andFessendon, (1982, Second Edition, Willard Grant Press, Boston, Mass.);Advanced Organic Chemistry by March (Third Edition, 1985, Wiley andSons, New York); and Advanced Organic Chemistry by Carey and Sundberg(Third Edition, Parts A and B, 1990, Plenum Press, New York). See, also,U.S. Patent Application Publications 2003/0082575 and 2003/0108885,which is incorporated by reference herein. In addition to unnaturalamino acids that contain novel side chains, unnatural amino acids thatmay be suitable for use in the present invention also optionallycomprise modified backbone structures, including but not limited to, asillustrated by the structures of Formula II and III:

wherein Z typically comprises OH, NH₂, SH, NH—R′, or S—R′, X and Y,which can be the same or different, typically comprise S or O, and R andR′, which are optionally the same or different, are typically selectedfrom the same list of constituents for the R group described above forthe unnatural amino acids having Formula I as well as hydrogen. Forexample, unnatural amino acids of the invention optionally comprisesubstitutions in the amino or carboxyl group as illustrated by FormulasII and III. Unnatural amino acids of this type include, but are notlimited to, α-hydroxy acids, α-thioacids, α-aminothiocarboxylates,including but not limited to, with side chains corresponding to thecommon twenty natural amino acids or unnatural side chains. In addition,substitutions at the α-carbon optionally include, but are not limitedto, L, D, or α-α-disubstituted amino acids such as D-glutamate,D-alanine, D-methyl-O-tyrosine, aminobutyric acid, and the like. Otherstructural alternatives include cyclic amino acids, such as prolineanalogues as well as 3, 4, 6, 7, 8, and 9 membered ring prolineanalogues, P and y amino acids such as substituted β-alanine and γ-aminobutyric acid.

Many unnatural amino acids are based on natural amino acids, such astyrosine, glutamine, phenylalanine, and the like, and are suitable foruse in the present invention. Tyrosine analogs include, but are notlimited to, para-substituted tyrosines, ortho-substituted tyrosines, andmeta substituted tyrosines, where the substituted tyrosine comprises,including but not limited to, a keto group (including but not limitedto, an acetyl group), a benzoyl group, an amino group, a hydrazine, anhydroxyamine, a thiol group, a carboxy group, an isopropyl group, amethyl group, a C₆-C₂₀ straight chain or branched hydrocarbon, asaturated or unsaturated hydrocarbon, an O-methyl group, a polyethergroup, a nitro group, an alkynyl group or the like. In addition,multiply substituted aryl rings are also contemplated. Glutamine analogsthat may be suitable for use in the present invention include, but arenot limited to, α-hydroxy derivatives, γ-substituted derivatives, cyclicderivatives, and amide substituted glutamine derivatives. Examplephenylalanine analogs that may be suitable for use in the presentinvention include, but are not limited to, para-substitutedphenylalanines, ortho-substituted phenyalanines, and meta-substitutedphenylalanines, where the substituent comprises, including but notlimited to, a hydroxy group, a methoxy group, a methyl group, an allylgroup, an aldehyde, an azido, an iodo, a bromo, a keto group (includingbut not limited to, an acetyl group), a benzoyl, an alkynyl group, orthe like. Specific examples of unnatural amino acids that may besuitable for use in the present invention include, but are not limitedto, a p-acetyl-L-phenylalanine, an O-methyl-L-tyrosine, anL-3-(2-naphthyl)alanine, a 3-methyl-phenylalanine, anO-4-allyl-L-tyrosine, a 4-propyl-L-tyrosine, atri-O-acetyl-GlcNAcβ-serine, an L-Dopa, a fluorinated phenylalanine, anisopropyl-L-phenylalanine, a p-azido-L-phenylalanine, ap-acyl-L-phenylalanine, a p-benzoyl-L-phenylalanine, an L-phosphoserine,a phosphonoserine, a phosphonotyrosine, a p-iodo-phenylalanine, ap-bromophenylalanine, a p-amino-L-phenylalanine, anisopropyl-L-phenylalanine, and a p-propargyloxy-phenylalanine, and thelike. Examples of structures of a variety of unnatural amino acids thatmay be suitable for use in the present invention are provided in, forexample, WO 2002/085923 entitled “In vivo incorporation of unnaturalamino acids.” See also Kiick et al., (2002) Incorporation of azides intorecombinant proteins for chemoselective modification by the Staudingerligation, PNAS 99:19-24, for additional methionine analogs.

Many of the unnatural amino acids suitable for use in the presentinvention are commercially available, e.g., from Sigma (USA) or Aldrich(Milwaukee, Wis., USA). Those that are not commercially available areoptionally synthesized as provided herein or as provided in variouspublications or using standard methods known to those of skill in theart. For organic synthesis techniques, see, e.g., Organic Chemistry byFessendon and Fessendon, (1982, Second Edition, Willard Grant Press,Boston Mass.); Advanced Organic Chemistry by March (Third Edition, 1985,Wiley and Sons, New York); and Advanced Organic Chemistry by Carey andSundberg (Third Edition, Parts A and B, 1990, Plenum Press, New York).Additional publications describing the synthesis of unnatural aminoacids include, e.g., WO 2002/085923 entitled “In vivo incorporation ofUnnatural Amino Acids;” Matsoukas et al., (1995) J. Med. Chem., 38,4660-4669; King, F. E. & Kidd, D. A. A. (1949) A New Synthesis ofGlutamine and of γ-Dipeptides of Glutamic Acid from PhthylatedIntermediates. J. Chem. Soc., 3315-3319; Friedman, O. M. & Chatterrji,R. (1959) Synthesis of Derivatives of Glutamine as Model Substrates forAnti-Tumor Agents. J. Am. Chem. Soc. 81, 3750-3752; Craig, J. C. et al.(1988) Absolute Configuration of the Enantiomers of 7-Chloro-4[[4-(diethylamino)-1-methylbutyl]amino]quinoline (Chloroquine). J. Org.Chem. 53, 1167-1170; Azoulay, M., Vilmont, M. & Frappier, F. (1991)Glutamine analogues as Potential Antimalarials, Eur. J. Med. Chem. 26,201-5; Koskinen, A. M. P. & Rapoport, H. (1989) Synthesis of4-Substituted Prolines as Conformationally Constrained Amino AcidAnalogues. J. Org. Chem. 54, 1859-1866; Christie, B. D. & Rapoport, H.(1985) Synthesis of Optically Pure Pipecolates from L-Asparagine.Application to the Total Synthesis of (+)-Apovincamine through AminoAcid Decarbonylation and Iminium Ion Cyclization. J. Org. Chem.1989:1859-1866; Barton et al., (1987) Synthesis of Novel a-Amino-Acidsand Derivatives Using Radical Chemistry Synthesis of L- andD-a-Amino-Adipic Acids, L-a-aminopimelic Acid and AppropriateUnsaturated Derivatives. Tetrahedron Lett. 43:4297-4308; and, Subasingheet al., (1992) Quisqualic acid analogues: synthesis of beta-heterocyclic2-aminopropanoic acid derivatives and their activity at a novelquisqualate-sensitized site. J. Med. Chem. 35:4602-7. See also, patentapplications entitled “Protein Arrays,” filed Dec. 22, 2003, Ser. No.10/744,899 and Ser. No. 60/435,821 filed on Dec. 22, 2002.

Amino acids with a carbonyl reactive group allow for a variety ofreactions to link molecules (including but not limited to, PEG or otherwater soluble molecules) via nucleophilic addition or aldol condensationreactions among others.

Exemplary carbonyl-containing amino acids can be represented as follows:

wherein n is 0-10; R₁ is an alkyl, aryl, substituted alkyl, orsubstituted aryl; R₂ is H, alkyl, aryl, substituted alkyl, andsubstituted aryl; and R₃ is H, an amino acid, a polypeptide, or an aminoterminus modification group, and R₄ is H, an amino acid, a polypeptide,or a carboxy terminus modification group. In some embodiments, n is 1,R₁ is phenyl and R₂ is a simple alkyl (i.e., methyl, ethyl, or propyl)and the ketone moiety is positioned in the para position relative to thealkyl side chain. In some embodiments, n is 1, R₁ is phenyl and R₂ is asimple alkyl (i.e., methyl, ethyl, or propyl) and the ketone moiety ispositioned in the meta position relative to the alkyl side chain.

In the present invention, a non-naturally encoded amino acid bearingadjacent hydroxyl and amino groups can be incorporated into thepolypeptide as a “masked” aldehyde functionality. For example,5-hydroxylysine bears a hydroxyl group adjacent to the epsilon amine.Reaction conditions for generating the aldehyde typically involveaddition of molar excess of sodium metaperiodate under mild conditionsto avoid oxidation at other sites within the polypeptide. The pH of theoxidation reaction is typically about 7.0. A typical reaction involvesthe addition of about 1.5 molar excess of sodium meta periodate to abuffered solution of the polypeptide, followed by incubation for about10 minutes in the dark. See, e.g. U.S. Pat. No. 6,423,685, which isincorporated by reference herein.

The carbonyl functionality can be reacted selectively with a hydrazine-,hydrazide-, hydroxylamine-, or semicarbazide-containing reagent undermild conditions in aqueous solution to form the corresponding hydrazone,oxime, or semicarbazone linkages, respectively, that are stable underphysiological conditions. See, e.g., Jencks, W. P., J. Am. Chem. Soc.81, 475-481 (1959); Shao, J. and Tam, J. P., J. Am. Chem. Soc.117:3893-3899 (1995). Moreover, the unique reactivity of the carbonylgroup allows for selective modification in the presence of the otheramino acid side chains. See, e.g., Cornish, V. W., et al., J. Am. Chem.Soc. 118:8150-8151 (1996); Geoghegan, K. F. & Stroh, J. G., Bioconjug.Chem. 3:138-146 (1992); Mahal, L. K., et al., Science 276:1125-1128(1997).

Non-naturally encoded amino acids containing a nucleophilic group, suchas a hydrazine, hydrazide or semicarbazide, allow for reaction with avariety of electrophilic groups to form conjugates (including but notlimited to, with PEG or other water soluble polymers).

Exemplary hydrazine, hydrazide or semicarbazide-containing amino acidscan be represented as follows:

wherein n is 0-10; R₁ is an alkyl, aryl, substituted alkyl, orsubstituted aryl or not present; X, is O, N, or S or not present; R₂ isH, an amino acid, a polypeptide, or an amino terminus modificationgroup, and R₃ is H, an amino acid, a polypeptide, or a carboxy terminusmodification group.

In some embodiments, n is 4, R₁ is not present, and X is N. In someembodiments, n is 2, R₁ is not present, and X is not present. In someembodiments, n is 1, R₁ is phenyl, X is O, and the oxygen atom ispositioned para to the alphatic group on the aryl ring.

Hydrazide-, hydrazine-, and semicarbazide-containing amino acids areavailable from commercial sources. For instance, L-glutamate-γ-hydrazideis available from Sigma Chemical (St. Louis, Mo.). Other amino acids notavailable commercially can be prepared by one skilled in the art. See,e.g., U.S. Pat. No. 6,281,211, which is incorporated by referenceherein.

Polypeptides containing non-naturally encoded amino acids that bearhydrazide, hydrazine or semicarbazide functionalities can be reactedefficiently and selectively with a variety of molecules that containaldehydes or other functional groups with similar chemical reactivity.See, e.g., Shao, J. and Tam, J., J. Am. Chem. Soc. 117:3893-3899 (1995).The unique reactivity of hydrazide, hydrazine and semicarbazidefunctional groups makes them significantly more reactive towardaldehydes, ketones and other electrophilic groups as compared to thenucleophilic groups present on the 20 common amino acids (including butnot limited to, the hydroxyl group of serine or threonine or the aminogroups of lysine and the N-terminus).

Non-naturally encoded amino acids containing an aminooxy (also called ahydroxylamine) group allow for reaction with a variety of electrophilicgroups to form conjugates (including but not limited to, with PEG orother water soluble polymers). Like hydrazines, hydrazides andsemicarbazides, the enhanced nucleophilicity of the aminooxy grouppermits it to react efficiently and selectively with a variety ofmolecules that contain aldehydes or other functional groups with similarchemical reactivity. See, e.g., Shao, J. and Tam, J., J. Am. Chem. Soc.117:3893-3899 (1995); H. Hang and C. Bertozzi, Acc. Chem. Res. 34:727-736 (2001). Whereas the result of reaction with a hydrazine group isthe corresponding hydrazone, however, an oxime results generally fromthe reaction of an aminooxy group with a carbonyl-containing group suchas a ketone.

Exemplary amino acids containing aminooxy groups can be represented asfollows:

wherein n is 0-10; R₁ is an alkyl, aryl, substituted alkyl, orsubstituted aryl or not present; X is O, N, S or not present; m is 0-10;Y═C(O) or not present; R₂ is H, an amino acid, a polypeptide, or anamino terminus modification group, and R₃ is H, an amino acid, apolypeptide, or a carboxy terminus modification group. In someembodiments, n is 1, R₁ is phenyl, X is O, m is 1, and Y is present. Insome embodiments, n is 2, R₁ and X are not present, m is 0, and Y is notpresent.

Aminooxy-containing amino acids can be prepared from readily availableamino acid precursors (homoserine, serine and threonine). See, e.g., M.Carrasco and R. Brown, J. Org. Chem. 68: 8853-8858 (2003). Certainaminooxy-containing amino acids, such as L-2-amino-4-(aminooxy)butyricacid), have been isolated from natural sources (Rosenthal, G. et al.,Life Sci. 60: 1635-1641 (1997). Other aminooxy-containing amino acidscan be prepared by one skilled in the art.

The unique reactivity of azide and alkyne functional groups makes themextremely useful for the selective modification of polypeptides andother biological molecules. Organic azides, particularly alphaticazides, and alkynes are generally stable toward common reactive chemicalconditions. In particular, both the azide and the alkyne functionalgroups are inert toward the side chains (i.e., R groups) of the 20common amino acids found in naturally-occurring polypeptides. Whenbrought into close proximity, however, the “spring-loaded” nature of theazide and alkyne groups is revealed and they react selectively andefficiently via Huisgen [3+2] cycloaddition reaction to generate thecorresponding triazole. See, e.g., Chin J., et al., Science 301:964-7(2003); Wang, Q., et al., J. Am. Chem. Soc. 125, 3192-3193 (2003); Chin,J. W., et al., J. Am. Chem. Soc. 124:9026-9027 (2002).

Because the Huisgen cycloaddition reaction involves a selectivecycloaddition reaction (see, e.g., Padwa, A., in COMPREHENSIVE ORGANICSYNTHESIS, Vol. 4, (ed. Trost, B. M., 1991), p. 1069-1109; Huisgen, R.in 1,3-DIPOLAR CYCLOADDITION CHEMISTRY, (ed. Padwa, A., 1984), p. 1-176)rather than a nucleophilic substitution, the incorporation ofnon-naturally encoded amino acids bearing azide and alkyne-containingside chains permits the resultant polypeptides to be modifiedselectively at the position of the non-naturally encoded amino acid.Cycloaddition reaction involving azide or alkyne-containing antibody canbe carried out at room temperature under aqueous conditions by theaddition of Cu(II) (including but not limited to, in the form of acatalytic amount of CuSO₄) in the presence of a reducing agent forreducing Cu(II) to Cu(I), in situ, in catalytic amount. See, e.g., Wang,Q., et al., J. Am. Chem. Soc. 125, 3192-3193 (2003); Tornoe, C. W., etal., J. Org. Chem. 67:3057-3064 (2002); Rostovtsev, et al., Angew. Chem.Int. Ed. 41:2596-2599 (2002). Exemplary reducing agents include,including but not limited to, ascorbate, metallic copper, quinine,hydroquinone, vitamin K, glutathione, cysteine, Fe²⁺, Co²⁺, and anapplied electric potential.

In some cases, where a Huisgen [3+2] cycloaddition reaction between anazide and an alkyne is desired, the antigen-binding polypeptidecomprises a non-naturally encoded amino acid comprising an alkyne moietyand the water soluble polymer to be attached to the amino acid comprisesan azide moiety. Alternatively, the converse reaction (i.e., with theazide moiety on the amino acid and the alkyne moiety present on thewater soluble polymer) can also be performed.

The azide functional group can also be reacted selectively with a watersoluble polymer containing an aryl ester and appropriatelyfunctionalized with an aryl phosphine moiety to generate an amidelinkage. The aryl phosphine group reduces the azide in situ and theresulting amine then reacts efficiently with a proximal ester linkage togenerate the corresponding amide. See, e.g., E. Saxon and C. Bertozzi,Science 287, 2007-2010 (2000). The azide-containing amino acid can beeither an alkyl azide (including but not limited to,2-amino-6-azido-1-hexanoic acid) or an aryl azide(p-azido-phenylalanine)

Exemplary water soluble polymers containing an aryl ester and aphosphine moiety can be represented as follows:

wherein X can be O, N, S or not present, Ph is phenyl, W is a watersoluble polymer and R can be H, alkyl, aryl, substituted alkyl andsubstituted aryl groups. Exemplary R groups include but are not limitedto —CH₂, —C(CH₃)₃, —OR′, —NR′R″, —SR′, -halogen, —C(O)R′, —CONR′R″,—S(O)₂R′, —S(O)₂NR′R″, —CN and —NO₂. R′, R″, R′″ and R″″ eachindependently refer to hydrogen, substituted or unsubstitutedheteroalkyl, substituted or unsubstituted aryl, including but notlimited to, aryl substituted with 1-3 halogens, substituted orunsubstituted alkyl, alkoxy or thioalkoxy groups, or arylalkyl groups.When a compound of the invention includes more than one R group, forexample, each of the R groups is independently selected as are each R′,R″, R′″ and R″″ groups when more than one of these groups is present.When R′ and R″ are attached to the same nitrogen atom, they can becombined with the nitrogen atom to form a 5-, 6-, or 7-membered ring.For example, —NR′R″ is meant to include, but not be limited to,1-pyrrolidinyl and 4-morpholinyl. From the above discussion ofsubstituents, one of skill in the art will understand that the term“alkyl” is meant to include groups including carbon atoms bound togroups other than hydrogen groups, such as haloalkyl (including but notlimited to, —CF₃ and —CH₂CF₃) and acyl (including but not limited to,—C(O)CH₃, —C(O)CF₃, —C(O)CH₂OCH₃, and the like).

The azide functional group can also be reacted selectively with a watersoluble polymer containing a thioester and appropriately functionalizedwith an aryl phosphine moiety to generate an amide linkage. The arylphosphine group reduces the azide in situ and the resulting amine thenreacts efficiently with the thioester linkage to generate thecorresponding amide. Exemplary water soluble polymers containing athioester and a phosphine moiety can be represented as follows:

wherein n is 1-10; X can be O, N, S or not present, Ph is phenyl, and Wis a water soluble polymer.

Exemplary alkyne-containing amino acids can be represented as follows:

wherein n is 0-10; R₁ is an alkyl, aryl, substituted alkyl, orsubstituted aryl or not present; X is O, N, S or not present; m is 0-10,R₂ is H, an amino acid, a polypeptide, or an amino terminus modificationgroup, and R₃ is H, an amino acid, a polypeptide, or a carboxy terminusmodification group. In some embodiments, n is 1, R₁ is phenyl, X is notpresent, m is 0 and the acetylene moiety is positioned in the paraposition relative to the alkyl side chain. In some embodiments, n is 1,R₁ is phenyl, X is O, m is 1 and the propargyloxy group is positioned inthe para position relative to the alkyl side chain (i.e.,O-propargyl-tyrosine). In some embodiments, n is 1, R₁ and X are notpresent and m is 0 (i.e., proparylglycine).

Alkyne-containing amino acids are commercially available. For example,propargylglycine is commercially available from Peptech (Burlington,Mass.). Alternatively, alkyne-containing amino acids can be preparedaccording to standard methods. For instance, p-propargyloxyphenylalaninecan be synthesized, for example, as described in Deiters, A., et al., J.Am. Chem. Soc. 125: 11782-11783 (2003), and 4-alkynyl-L-phenylalaninecan be synthesized as described in Kayser, B., et al., Tetrahedron53(7): 2475-2484 (1997). Other alkyne-containing amino acids can beprepared by one skilled in the art.

Exemplary azide-containing amino acids can be represented as follows:

wherein n is 0-10; R₁ is an alkyl, aryl, substituted alkyl, substitutedaryl or not present; X is O, N, S or not present; m is 0-10; R₂ is H, anamino acid, a polypeptide, or an amino terminus modification group, andR₃ is H, an amino acid, a polypeptide, or a carboxy terminusmodification group. In some embodiments, n is 1, R₁ is phenyl, X is notpresent, m is 0 and the azide moiety is positioned para to the alkylside chain. In some embodiments, n is 0-4 and R₁ and X are not present,and m=0. In some embodiments, n is 1, R₁ is phenyl, X is O, m is 2 andthe P-azidoethoxy moiety is positioned in the para position relative tothe alkyl side chain.

Azide-containing amino acids are available from commercial sources. Forinstance, 4-azidophenylalanine can be obtained from Chem-ImpexInternational, Inc. (Wood Dale, Ill.). For those azide-containing aminoacids that are not commercially available, the azide group can beprepared relatively readily using standard methods known to those ofskill in the art, including but not limited to, via displacement of asuitable leaving group (including but not limited to, halide, mesylate,tosylate) or via opening of a suitably protected lactone. See, e.g.,Advanced Organic Chemistry by March (Third Edition, 1985, Wiley andSons, New York).

The unique reactivity of beta-substituted aminothiol functional groupsmakes them extremely useful for the selective modification ofpolypeptides and other biological molecules that contain aldehyde groupsvia formation of the thiazolidine. See, e.g., J. Shao and J. Tam, J. Am.Chem. Soc. 1995, 117 (14) 3893-3899. In some embodiments,beta-substituted aminothiol amino acids can be incorporated intoantibodies and then reacted with water soluble polymers comprising analdehyde functionality. In some embodiments, a water soluble polymer,drug conjugate or other payload can be coupled to an antibodypolypeptide comprising a beta-substituted aminothiol amino acid viaformation of the thiazolidine.

Particular examples of useful non-natural amino acids include, but arenot limited to, p-acetyl-L-phenylalanine, O-methyl-L-tyrosine,L-3-(2-naphthyl)alanine, 3-methyl-phenylalanine, O-4-allyl-L-tyrosine,4-propyl-L-tyrosine, tri-O-acetyl-GlcNAc b-serine, L-Dopa, fluorinatedphenylalanine, isopropyl-L-phenylalanine, p-azido-L-phenylalanine,p-acyl-L-phenylalanine, p-benzoyl-L-phenylalanine, L-phosphoserine,phosphonoserine, phosphonotyrosine, p-iodo-phenylalanine,p-bromophenylalanine, p-amino-L-phenylalanine,isopropyl-L-phenylalanine, and p-propargyloxy-phenylalanine. Furtheruseful examples include N-acetyl-L-glucosaminyl-L-serine,N-acetyl-L-galactosaminyl-L-serine, N-acetyl-L-glucosaminyl-L-threonine,N-acetyl-L-glucosaminyl-L-asparagine and O-mannosaminyl-L-serine.

In particular embodiments, the non-natural amino acids are selected fromp-acetyl-phenylalanine, p-ethynyl-phenylalanine,p-propargyloxyphenylalanine, and p-azido-phenylalanine. One particularlyuseful non-natural amino acid is p-azido phenylalanine. This amino acidresidue is known to those of skill in the art to facilitate Huisgen[3+2] cyloaddition reactions (so-called “click” chemistry reactions)with, for example, compounds bearing alkynyl groups. This reactionenables one of skill in the art to readily and rapidly conjugate to theantibody at the site-specific location of the non-natural amino acid.

In certain embodiments, the first reactive group is an alkynyl moiety(including but not limited to, in the unnatural amino acidp-propargyloxyphenylalanine, where the propargyl group is also sometimesreferred to as an acetylene moiety) and the second reactive group is anazido moiety, and [3+2] cycloaddition chemistry can be used. In certainembodiments, the first reactive group is the azido moiety (including butnot limited to, in the unnatural amino acid p-azido-L-phenylalanine) andthe second reactive group is the alkynyl moiety.

In the above formulas, each L represents a divalent linker. The divalentlinker can be any divalent linker known to those of skill in the art.Generally, the divalent linker is capable of forming covalent bonds tothe functional moiety R and the alpha carbon of the non-natural aminoacid. Useful divalent linkers a bond, alkylene, substituted alkylene,heteroalkylene, substituted heteroalkylene, arylene, substitutedarylene, heteroarlyene and substituted heteroarylene. In certainembodiments, L is C₁₋₁₀ alkylene or C₁₋₁₀ heteroalkylene.

The non-natural amino acids used in the methods and compositionsdescribed herein have at least one of the following four properties: (1)at least one functional group on the sidechain of the non-natural aminoacid has at least one characteristics and/or activity and/or reactivityorthogonal to the chemical reactivity of the 20 common,genetically-encoded amino acids (i.e., alanine, arginine, asparagine,aspartic acid, cysteine, glutamine, glutamic acid, glycine, histidine,isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine,threonine, tryptophan, tyrosine, and valine), or at least orthogonal tothe chemical reactivity of the naturally occurring amino acids presentin the polypeptide that includes the non-natural amino acid; (2) theintroduced non-natural amino acids are substantially chemically inerttoward the 20 common, genetically-encoded amino acids; (3) thenon-natural amino acid can be stably incorporated into a polypeptide,preferably with the stability commensurate with the naturally-occurringamino acids or under typical physiological conditions, and furtherpreferably such incorporation can occur via an in vivo system; and (4)the non-natural amino acid includes an oxime functional group or afunctional group that can be transformed into an oxime group by reactingwith a reagent, preferably under conditions that do not destroy thebiological properties of the polypeptide that includes the non-naturalamino acid (unless of course such a destruction of biological propertiesis the purpose of the modification/transformation), or where thetransformation can occur under aqueous conditions at a pH between about4 and about 8, or where the reactive site on the non-natural amino acidis an electrophilic site. Illustrative, non-limiting examples of aminoacids that satisfy these four properties for non-natural amino acidsthat can be used with the compositions and methods described herein arepresented in FIGS. 2, 3, 35 and 40-43. Any number of non-natural aminoacids can be introduced into the polypeptide. Non-natural amino acidsmay also include protected or masked oximes or protected or maskedgroups that can be transformed into an oxime group after deprotection ofthe protected group or unmasking of the masked group. Non-natural aminoacids may also include protected or masked carbonyl or dicarbonylgroups, which can be transformed into a carbonyl or dicarbonyl groupafter deprotection of the protected group or unmasking of the maskedgroup and thereby are available to react with hydroxylamines or oximesto form oxime groups.

In further embodiments, non-natural amino acids that may be used in themethods and compositions described herein include, but are not limitedto, amino acids comprising a photoactivatable cross-linker, spin-labeledamino acids, fluorescent amino acids, metal binding amino acids,metal-containing amino acids, radioactive amino acids, amino acids withnovel functional groups, amino acids that covalently or non-covalentlyinteract with other molecules, photocaged and/or photoisomerizable aminoacids, amino acids comprising biotin or a biotin analogue, glycosylatedamino acids such as a sugar substituted serine, other carbohydratemodified amino acids, keto-containing amino acids, aldehyde-containingamino acids, amino acids comprising polyethylene glycol or otherpolyethers, heavy atom substituted amino acids, chemically cleavableand/or photocleavable amino acids, amino acids with an elongated sidechains as compared to natural amino acids, including but not limited to,polyethers or long chain hydrocarbons, including but not limited to,greater than about 5 or greater than about 10 carbons, carbon-linkedsugar-containing amino acids, redox-active amino acids, amino thioacidcontaining amino acids, and amino acids comprising one or more toxicmoiety.

In some embodiments, non-natural amino acids comprise a saccharidemoiety. Examples of such amino acids includeN-acetyl-L-glucosaminyl-L-serine, N-acetyl-L-galactosaminyl-L-serine,N-acetyl-L-glucosaminyl-L-threonine,N-acetyl-L-glucosaminyl-L-asparagine and O-mannosaminyl-L-serine.Examples of such amino acids also include examples where thenaturally-occurring N- or O-linkage between the amino acid and thesaccharide is replaced by a covalent linkage not commonly found innature—including but not limited to, an alkene, an oxime, a thioether,an amide and the like. Examples of such amino acids also includesaccharides that are not commonly found in naturally-occurring proteinssuch as 2-deoxy-glucose, 2-deoxygalactose and the like.

The chemical moieties incorporated into antibodies via incorporation ofnon-natural amino acids offer a variety of advantages and manipulationsof polypeptides. For example, the unique reactivity of a carbonyl ordicarbonyl functional group (including a keto- or aldehyde-functionalgroup) allows selective modification of antibodies with any of a numberof hydrazine- or hydroxylamine-containing reagents in vivo and in vitro.A heavy atom non-natural amino acid, for example, can be useful forphasing x-ray structure data. The site-specific introduction of heavyatoms using non-natural amino acids also provides selectivity andflexibility in choosing positions for heavy atoms. Photoreactivenon-natural amino acids (including but not limited to, amino acids withbenzophenone and arylazides (including but not limited to, phenylazide)side chains), for example, allow for efficient in vivo and in vitrophotocrosslinking of polypeptides. Examples of photoreactive non-naturalamino acids include, but are not limited to, p-azido-phenylalanine andp-benzoyl-phenylalanine. The antibodies with the photoreactivenon-natural amino acids may then be crosslinked at will by excitation ofthe photoreactive group-providing temporal control. In a non-limitingexample, the methyl group of a non-natural amino can be substituted withan isotopically labeled, including but not limited to, with a methylgroup, as a probe of local structure and dynamics, including but notlimited to, with the use of nuclear magnetic resonance and vibrationalspectroscopy.

Amino acids with an electrophilic reactive group allow for a variety ofreactions to link molecules via various chemical reactions, including,but not limited to, nucleophilic addition reactions. Such electrophilicreactive groups include a carbonyl- or dicarbonyl-group (including aketo- or aldehyde group), a carbonyl-like- or dicarbonyl-like-group(which has reactivity similar to a carbonyl- or dicarbonyl-group and isstructurally similar to a carbonyl- or dicarbonyl-group), a maskedcarbonyl- or masked dicarbonyl-group (which can be readily convertedinto a carbonyl- or dicarbonyl-group), or a protected carbonyl- orprotected dicarbonyl-group (which has reactivity similar to a carbonyl-or dicarbonyl-group upon deprotection). Such amino acids include aminoacids having the structure of Formula (I):

wherein: A is optional, and when present is lower alkylene, substitutedlower alkylene, lower cycloalkylene, substituted lower cycloalkylene,lower alkenylene, substituted lower alkenylene, alkynylene, lowerheteroalkylene, substituted heteroalkylene, lower heterocycloalkylene,substituted lower heterocycloalkylene, arylene, substituted arylene,heteroarylene, substituted heteroarylene, alkarylene, substitutedalkarylene, aralkylene, or substituted aralkylene; B is optional, andwhen present is a linker selected from the group consisting of loweralkylene, substituted lower alkylene, lower alkenylene, substitutedlower alkenylene, lower heteroalkylene, substituted lowerheteroalkylene, —O—, —O-(alkylene or substituted alkylene)-, —S—,—S-(alkylene or substituted alkylene)-, —S(O)_(k)— where k is 1, 2, or3, —S(O)_(k)(alkylene or substituted alkylene)-, —C(O)—, —NS(O)₂—,—OS(O)₂—, —C(O)-(alkylene or substituted alkylene)-, —C(S)—,—C(S)-(alkylene or substituted alkylene)-, —N(R′)—, —NR′-(alkylene orsubstituted alkylene)-, —C(O)N(R′)—, —CON(R′)-(alkylene or substitutedalkylene)-, —CSN(R′)—, —CSN(R′)-(alkylene or substituted alkylene)-,—N(R′)CO-(alkylene or substituted alkylene)-, —N(R′)C(O)O—,—S(O)_(k)N(R′)—, —N(R′)C(O)N(R′)—, —N(R′)C(S)N(R′)—,—N(R′)S(O)_(k)N(R′)—, —N(R′)—N═, —C(R′)═N—, —C(R′)═N—N(R′)—,—C(R′)═N—N═, —C(R′)₂—N═N—, and —C(R′)₂—N(R′)—N(R′)—, where each R′ isindependently H, alkyl, or substituted alkyl; J is

R is H, alkyl, substituted alkyl, cycloalkyl, or substituted cycloalkyl;each R″ is independently H, alkyl, substituted alkyl, or a protectinggroup, or when more than one R″ group is present, two R″ optionally forma heterocycloalkyl; R₁ is H, an amino protecting group, resin, aminoacid, polypeptide, or polynucleotide; and R₂ is OH, an ester protectinggroup, resin, amino acid, polypeptide, or polynucleotide; each of R₃ andR₄ is independently H, halogen, lower alkyl, or substituted lower alkyl,or R₃ and R₄ or two R₃ groups optionally form a cycloalkyl or aheterocycloalkyl; or the -A-B-J-R groups together form a bicyclic ortricyclic cycloalkyl or heterocycloalkyl comprising at least onecarbonyl group, including a dicarbonyl group, protected carbonyl group,including a protected dicarbonyl group, or masked carbonyl group,including a masked dicarbonyl group; or the -J-R group together forms amonocyclic or bicyclic cycloalkyl or heterocycloalkyl comprising atleast one carbonyl group, including a dicarbonyl group, protectedcarbonyl group, including a protected dicarbonyl group, or maskedcarbonyl group, including a masked dicarbonyl group; with a proviso thatwhen A is phenylene and each R₃ is H, B is present; and that when A is—(CH₂)₄— and each R₃ is H, B is not —NHC(O)(CH₂CH₂)—; and that when Aand B are absent and each R₃ is H, R is not methyl. Such non-naturalamino acids may be in the form of a salt, or may be incorporated into anon-natural amino acid polypeptide, polymer, polysaccharide, or apolynucleotide and optionally post translationally modified.

In certain embodiments, compounds of Formula (I) are stable in aqueoussolution for at least 1 month under mildly acidic conditions. In certainembodiments, compounds of Formula (I) are stable for at least 2 weeksunder mildly acidic conditions. In certain embodiments, compound ofFormula (I) are stable for at least 5 days under mildly acidicconditions. In certain embodiments, such acidic conditions are pH 2 to8.

In certain embodiments of compounds of Formula (I), B is lower alkylene,substituted lower alkylene, —O-(alkylene or substituted alkylene)-,—C(R′)═N—N(R′)—, —N(R′)CO—, —C(O)—, —C(R′)═N—, —C(O)-(alkylene orsubstituted alkylene)-, —CON(R′)-(alkylene or substituted alkylene)-,—S(alkylene or substituted alkylene)-, —S(O)(alkylene or substitutedalkylene)-, or —S(O)₂(alkylene or substituted alkylene)-. In certainembodiments of compounds of Formula (I), B is —O(CH₂)—, —CH═N—,—CH═N—NH—, —NHCH₂—, —NHCO—, —C(O)—, —C(O)—(CH₂)—, —CONH—(CH₂)—, —SCH₂—,—S(═O)CH₂—, or —S(O)₂CH₂—. In certain embodiments of compounds ofFormula (I), R is C₁-6 alkyl or cycloalkyl. In certain embodiments ofcompounds of Formula (I) R is —CH₃, —CH(CH₃)₂, or cyclopropyl. Incertain embodiments of compounds of Formula (I), R₁ is H,tert-butyloxycarbonyl (Boc), 9-Fluorenylmethoxycarbonyl (Fmoc),N-acetyl, tetrafluoroacetyl (TFA), or benzyloxycarbonyl (Cbz). Incertain embodiments of compounds of Formula (I), R₁ is a resin, aminoacid, polypeptide, or polynucleotide. In certain embodiments ofcompounds of Formula (I), R₂ is OH, O-methyl, O-ethyl, or O-t-butyl. Incertain embodiments of compounds of Formula (I), R₂ is a resin, aminoacid, polypeptide, or polynucleotide. In certain embodiments ofcompounds of Formula (I), R₂ is a polynucleotide. In certain embodimentsof compounds of Formula (I), R₂ is ribonucleic acid (RNA). In certainembodiments of compounds of Formula (I), R₂ is tRNA. In certainembodiments of compounds of Formula (I), the tRNA specificallyrecognizes a selector codon. In certain embodiments of compounds ofFormula (I) the selector codon is selected from the group consisting ofan amber codon, ochre codon, opal codon, a unique codon, a rare codon,an unnatural codon, a five-base codon, and a four-base codon. In certainembodiments of compounds of Formula (I), R₂ is a suppressor tRNA.

In certain embodiments of compounds of Formula (I),

is selected from the group consisting of: (i) A is substituted loweralkylene, C₄-arylene, substituted arylene, heteroarylene, substitutedheteroarylene, alkarylene, substituted alkarylene, aralkylene, orsubstituted aralkylene; B is optional, and when present is a divalentlinker selected from the group consisting of lower alkylene, substitutedlower alkylene, lower alkenylene, substituted lower alkenylene, —O—,—O-(alkylene or substituted alkylene)-, —S—, —S(O)—, —S(O)₂N(R′) ,—NS(O)₂—, —OS(O)₂—, —C(O)—, —C(O)-(alkylene or substituted alkylene)-,—C(S)—, —N(R′)—, —C(O)N(R′)—, —CON(R′)-(alkylene or substitutedalkylene)-, —CSN(R′)—, —N(R′)CO-(alkylene or substituted alkylene)-,—N(R′)C(O)O—, —N(R′)C(S)—, —S(O)N(R′), —S(O)₂N(R′), —N(R′)C(O)N(R′)—,—N(R′)C(S)N(R′)—, —N(R′)S(O)N(R′)—, —N(R′)S(O)₂N(R′)—, —N(R′)—N═,—C(R′)═N—N(R′)—, —C(R′)═N—N═, —C(R′)₂—N═N—, and —C(R′)₂—N(R′)—N(R′)—;(ii) A is optional, and when present is substituted lower alkylene,C₄-arylene, substituted arylene, heteroarylene, substitutedheteroarylene, alkarylene, substituted alkarylene, aralkylene, orsubstituted aralkylene; B is a divalent linker selected from the groupconsisting of lower alkylene, substituted lower alkylene, loweralkenylene, substituted lower alkenylene, —O—, —O-(alkylene orsubstituted alkylene)-, —S—, —S(O)—, —S(O)₂—, —NS(O)₂—, —OS(O)₂—,—C(O)—, —C(O)-(alkylene or substituted alkylene)-, —C(S)—, —N(R′)—,—C(O)N(R′)—, —CON(R′)-(alkylene or substituted alkylene)-, —CSN(R′)—,—N(R′)CO-(alkylene or substituted alkylene)-, —N(R′)C(O)O—, —N(R′)C(S)—,—S(O)N(R′), —S(O)₂N(R′), —N(R′)C(O)N(R′)—, —N(R′)C(S)N(R′)—,—N(R′)S(O)N(R′)—, —N(R′)S(O)₂N(R′)—, —N(R′)—N═, —C(R′)═N—N(R′)—,—C(R′)═N—N═, —C(R′)₂—N═N—, and —C(R′)₂—N(R′)—N(R′)—; (iii) A is loweralkylene; B is optional, and when present is a divalent linker selectedfrom the group consisting of lower alkylene, substituted lower alkylene,lower alkenylene, substituted lower alkenylene, —O—, —O-(alkylene orsubstituted alkylene)-, —S—, —S(O)—, —S(O)₂—, —NS(O)₂—, —OS(O)₂—,—C(O)—, —C(O)-(alkylene or substituted alkylene)-, —C(S)—, —N(R′)—,—C(O)N(R′)—, —CSN(R′)—, —CON(R′)-(alkylene or substituted alkylene)-,—N(R′)C(O)O—, —N(R′)C(S)—, —S(O)N(R′), —S(O)₂N(R′), —N(R′)C(O)N(R′)—,—N(R′)C(S)N(R′)—, —N(R′)S(O)N(R′)—, —N(R′)S(O)₂N(R′)—, —N(R′)—N═,—C(R′)═N—N(R′)—, —C(R′)═N—N═, —C(R′)₂—N═N—, and —C(R′)₂—N(R′)—N(R′)—;and (iv) A is phenylene; B is a divalent linker selected from the groupconsisting of lower alkylene, substituted lower alkylene, loweralkenylene, substituted lower alkenylene, —O—, —O-(alkylene orsubstituted alkylene)-, —S—, —S(O)—, —S(O)₂—, —NS(O)₂—, —OS(O)₂—,—C(O)—, —C(O)-(alkylene or substituted alkylene)-, —C(S)—, —N(R′)—,—C(O)N(R′)—, —CON(R′)-(alkylene or substituted alkylene)-, —CSN(R′)—,—N(R′)CO-(alkylene or substituted alkylene)-, —N(R′)C(O)O—, —N(R′)C(S)—,—S(O)N(R′), —S(O)₂N(R′), —N(R′)C(O)N(R′)—, —N(R′)C(S)N(R′)—,—N(R′)S(O)N(R′)—, —N(R′)S(O)₂N(R′)—, —N(R′)—N═, —C(R′)′N—N(R′)—,—C(R′)═N—N═, —C(R′)₂—N═N—, and —C(R′)₂—N(R′)—N(R′)—; J is

each R′ is independently H, alkyl, or substituted alkyl; R₁ is optional,and when present, is H, an amino protecting group, resin, amino acid,polypeptide, or polynucleotide; and R₂ is optional, and when present, isOH, an ester protecting group, resin, amino acid, polypeptide, orpolynucleotide; and each R₃ and R₄ is independently H, halogen, loweralkyl, or substituted lower alkyl; and R is H, alkyl, substituted alkyl,cycloalkyl, or substituted cycloalkyl.

In addition, amino acids having the structure of Formula (II) areincluded:

wherein: A is optional, and when present is lower alkylene, substitutedlower alkylene, lower cycloalkylene, substituted lower cycloalkylene,lower alkenylene, substituted lower alkenylene, alkynylene, lowerheteroalkylene, substituted heteroalkylene, lower heterocycloalkylene,substituted lower heterocycloalkylene, arylene, substituted arylene,heteroarylene, substituted heteroarylene, alkarylene, substitutedalkarylene, aralkylene, or substituted aralkylene; B is optional, andwhen present is a linker selected from the group consisting of loweralkylene, substituted lower alkylene, lower alkenylene, substitutedlower alkenylene, lower heteroalkylene, substituted lowerheteroalkylene, —O—, —O-(alkylene or substituted alkylene)-, —S—,—S-(alkylene or substituted alkylene)-, —S(O)_(k)— where k is 1, 2, or3, —S(O)_(k)(alkylene or substituted alkylene)-, —C(O)—, —NS(O)₂—,—OS(O)₂—, —C(O)-(alkylene or substituted alkylene)-, —C(S)—,—C(S)-(alkylene or substituted alkylene)-, —N(R′)—, —NR′-(alkylene orsubstituted alkylene)-, —C(O)N(R′)—, —CON(R′)-(alkylene or substitutedalkylene)-, —CSN(R′)—, —CSN(R′)-(alkylene or substituted alkylene)-,—N(R′)CO-(alkylene or substituted alkylene)-, —N(R′)C(O)O—,—S(O)_(k)N(R′)—, —N(R′)C(O)N(R′)—, —N(R′)C(S)N(R′)—,—N(R′)S(O)_(k)N(R′)—, —N(R′)—N═, —C(R′)═N—, —C(R′)═N—N(R′)—,—C(R′)═N—N═, —C(R′)₂—N═N—, and —C(R′)₂—N(R′)—N(R′)—, where each R′ isindependently H, alkyl, or substituted alkyl; R is H, alkyl, substitutedalkyl, cycloalkyl, or substituted cycloalkyl; R₁ is H, an aminoprotecting group, resin, amino acid, polypeptide, or polynucleotide; andR₂ is OH, an ester protecting group, resin, amino acid, polypeptide, orpolynucleotide. In certain embodiments, when A is phenylene, B ispresent; and that when A is —(CH₂)₄—, B is not —NHC(O)(CH₂CH₂)—; andthat when A and B are absent, R is not methyl. Such non-natural aminoacids may be in the form of a salt, or may be incorporated into anon-natural amino acid polypeptide, polymer, polysaccharide, or apolynucleotide and optionally post translationally modified.

In addition, amino acids having the structure of Formula (III) areincluded:

wherein: B is a linker selected from the group consisting of loweralkylene, substituted lower alkylene, lower alkenylene, substitutedlower alkenylene, lower heteroalkylene, substituted lowerheteroalkylene, —O—, —O-(alkylene or substituted alkylene)-, —S—,—S-(alkylene or substituted alkylene)-, —S(O)_(k)— where k is 1, 2, or3, —S(O)_(k)(alkylene or substituted alkylene)-, —C(O)—, —NS(O)₂—,—OS(O)₂—, —C(O)-(alkylene or substituted alkylene)-, —C(S)—,—C(S)-(alkylene or substituted alkylene)-, —N(R′)—, —NR′-(alkylene orsubstituted alkylene)-, —C(O)N(R′)—, —CON(R′)-(alkylene or substitutedalkylene)-, —CSN(R′)—, —CSN(R′)-(alkylene or substituted alkylene)-,—N(R′)CO-(alkylene or substituted alkylene)-, —N(R′)C(O)O—,—S(O)_(k)N(R′)—, —N(R′)C(O)N(R′)—, —N(R′)C(S)N(R′)—,—N(R′)S(O)_(k)N(R′)—, —N(R′)—N═, —C(R′)═N—, —C(R′)═N—N(R′)—,—C(R′)═N—N═, —C(R′)₂—N═N—, and —C(R′)₂—N(R′)—N(R′)—, where each R′ isindependently H, alkyl, or substituted alkyl; R is H, alkyl, substitutedalkyl, cycloalkyl, or substituted cycloalkyl; R₁ is H, an aminoprotecting group, resin, amino acid, polypeptide, or polynucleotide; andR₂ is OH, an ester protecting group, resin, amino acid, polypeptide, orpolynucleotide; each R_(a) is independently selected from the groupconsisting of H, halogen, alkyl, substituted alkyl, —N(R′)₂, —C(O)_(k)R′where k is 1, 2, or 3, —C(O)N(R′)₂, —OR′, and —S(O)_(k)R′, where each R′is independently H, alkyl, or substituted alkyl. Such non-natural aminoacids may be in the form of a salt, or may be incorporated into anon-natural amino acid polypeptide, polymer, polysaccharide, or apolynucleotide and optionally post translationally modified.

In addition, the following amino acids are included:

Such non-natural amino acids may be are optionally amino protectedgroup, carboxyl protected and/or in the form of a salt, or may beincorporated into a non-natural amino acid polypeptide, polymer,polysaccharide, or a polynucleotide and optionally post translationallymodified.

In addition, the following amino acids having the structure of Formula(IV) are included:

wherein —NS(O)₂—, —OS(O)₂—, optional, and when present is a linkerselected from the group consisting of lower alkylene, substituted loweralkylene, lower alkenylene, substituted lower alkenylene, lowerheteroalkylene, substituted lower heteroalkylene, —O—, —O-(alkylene orsubstituted alkylene)-, —S—, —S-(alkylene or substituted alkylene)-,—S(O)_(k)— where k is 1, 2, or 3, —S(O)_(k)(alkylene or substitutedalkylene)-, —C(O)—, —C(O)-(alkylene or substituted alkylene)-, —C(S)—,—C(S)-(alkylene or substituted alkylene)-, —N(R′)—, —NR′-(alkylene orsubstituted alkylene)-, —C(O)N(R′)—, —CON(R′)-(alkylene or substitutedalkylene)-, —CSN(R′)—, —CSN(R′)-(alkylene or substituted alkylene)-,—N(R′)CO-(alkylene or substituted alkylene)-, —N(R′)C(O)O—,—S(O)_(k)N(R′)—, —N(R′)C(O)N(R′)—, —N(R′)C(S)N(R′)—,—N(R′)S(O)_(k)N(R′)—, —N(R′)—N═, —C(R′)═N—, —C(R′)═N—N(R′)—,—C(R′)═N—N═, —C(R′)₂—N═N—, and —C(R′)₂—N(R′)—N(R′)—, where each R′ isindependently H, alkyl, or substituted alkyl; R is H, alkyl, substitutedalkyl, cycloalkyl, or substituted cycloalkyl; R₁ is H, an aminoprotecting group, resin, amino acid, polypeptide, or polynucleotide; andR₂ is OH, an ester protecting group, resin, amino acid, polypeptide, orpolynucleotide; each R_(a) is independently selected from the groupconsisting of H, halogen, alkyl, substituted alkyl, —N(R′)₂, —C(O)_(k)R′where k is 1, 2, or 3, —C(O)N(R′)₂, —OR′, and —S(O)_(k)R′, where each R′is independently H, alkyl, or substituted alkyl; and n is 0 to 8. Incertain embodiments, when A is —(CH₂)₄—, B is not —NHC(O)(CH₂CH₂)—. Suchnon-natural amino acids may be in the form of a salt, or may beincorporated into a non-natural amino acid polypeptide, polymer,polysaccharide, or a polynucleotide and optionally post translationallymodified.

In addition, the following amino acids are included:

wherein such compounds are optionally amino protected, optionallycarboxyl protected, optionally amino protected and carboxyl protected,or a salt thereof, or may be incorporated into a non-natural amino acidpolypeptide, polymer, polysaccharide, or a polynucleotide and optionallypost translationally modified.

In addition, the following amino acids having the structure of Formula(VIII) are included:

wherein, A is optional, and when present is lower alkylene, substitutedlower alkylene, lower cycloalkylene, substituted lower cycloalkylene,lower alkenylene, substituted lower alkenylene, alkynylene, lowerheteroalkylene, substituted heteroalkylene, lower heterocycloalkylene,substituted lower heterocycloalkylene, arylene, substituted arylene,heteroarylene, substituted heteroarylene, alkarylene, substitutedalkarylene, aralkylene, or substituted aralkylene; B is optional, andwhen present is a linker selected from the group consisting of loweralkylene, substituted lower alkylene, lower alkenylene, substitutedlower alkenylene, lower heteroalkylene, substituted lowerheteroalkylene, —O—, —O-(alkylene or substituted alkylene)-, —S—,—S-(alkylene or substituted alkylene)-, —S(O)_(k)— where k is 1, 2, or3, —S(O)_(k)(alkylene or substituted alkylene)-, —C(O)—, —NS(O)₂—,—OS(O)₂—, —C(O)-(alkylene or substituted alkylene)-, —C(S)—,—C(S)-(alkylene or substituted alkylene)-, —N(R′)—, —NR′-(alkylene orsubstituted alkylene)-, —C(O)N(R′)—, —CON(R′)-(alkylene or substitutedalkylene)-, —CSN(R′)—, —CSN(R′)-(alkylene or substituted alkylene)-,—N(R′)CO-(alkylene or substituted alkylene)-, —N(R′)C(O)O—,—S(O)_(k)N(R′)—, —N(R′)C(O)N(R′)—, —N(R′)C(S)N(R′)—,—N(R′)S(O)_(k)N(R′)—, —N(R′)—N═, —C(R′)═N—, —C(R′)═N—N(R′)—,—C(R′)═N—N═, —C(R′)₂—N═N—, and —C(R′)₂—N(R′)—N(R′)—, where each R′ isindependently H, alkyl, or substituted alkyl; R₁ is H, an aminoprotecting group, resin, amino acid, polypeptide, or polynucleotide; andR₂ is OH, an ester protecting group, resin, amino acid, polypeptide, orpolynucleotide. Such non-natural amino acids may be in the form of asalt, or may be incorporated into a non-natural amino acid polypeptide,polymer, polysaccharide, or a polynucleotide and optionally posttranslationally modified.

In addition, the following amino acids having the structure of Formula(IX) are included:

wherein, B is optional, and when present is a linker selected from thegroup consisting of lower alkylene, substituted lower alkylene, loweralkenylene, substituted lower alkenylene, lower heteroalkylene,substituted lower heteroalkylene, —O—, —O-(alkylene or substitutedalkylene)-, —S—, —S-(alkylene or substituted alkylene)-, —S(O)_(k)—where k is 1, 2, or 3, —S(O)_(k)(alkylene or substituted alkylene)-,—C(O)—, —NS(O)₂—, —OS(O)₂—, —C(O)-(alkylene or substituted alkylene)-,—C(S)—, —C(S)-(alkylene or substituted alkylene)-, —N(R′)—,—NR′-(alkylene or substituted alkylene)-, —C(O)N(R′)—,—CON(R′)-(alkylene or substituted alkylene)-, —CSN(R′)—,—CSN(R′)-(alkylene or substituted alkylene)-, —N(R′)CO-(alkylene orsubstituted alkylene)-, —N(R′)C(O)O—, —S(O)_(k)N(R′)—, —N(R′)C(O)N(R′)—,—N(R′)C(S)N(R′)—, —N(R′)S(O)_(k)N(R′)—, —N(R′)—N═, —C(R′)═N—,—C(R′)═N—N(R′)—, —C(R′)═N—N═, —C(R′)₂—N═N—, and —C(R′)₂—N(R′)—N(R′)—,where each R′ is independently H, alkyl, or substituted alkyl; R is H,alkyl, substituted alkyl, cycloalkyl, or substituted cycloalkyl; R₁ isH, an amino protecting group, resin, amino acid, polypeptide, orpolynucleotide; and R₂ is OH, an ester protecting group, resin, aminoacid, polypeptide, or polynucleotide; wherein each R_(a) isindependently selected from the group consisting of H, halogen, alkyl,substituted alkyl, —N(R′)₂, —C(O)_(k)R′ where k is 1, 2, or 3,—C(O)N(R′)₂, —OR′, and —S(O)_(k)R′, where each R′ is independently H,alkyl, or substituted alkyl. Such non-natural amino acids may be in theform of a salt, or may be incorporated into a non-natural amino acidpolypeptide, polymer, polysaccharide, or a polynucleotide and optionallypost translationally modified.

In addition, the following amino acids are included:

wherein such compounds are optionally amino protected, optionallycarboxyl protected, optionally amino protected and carboxyl protected,or a salt thereof, or may be incorporated into a non-natural amino acidpolypeptide, polymer, polysaccharide, or a polynucleotide and optionallypost translationally modified.

In addition, the following amino acids having the structure of Formula(X) are included:

wherein, B is optional, and when present is a linker selected from thegroup consisting of lower alkylene, substituted lower alkylene, loweralkenylene, substituted lower alkenylene, lower heteroalkylene,substituted lower heteroalkylene, —O—, —O-(alkylene or substitutedalkylene)-, —S—, —S-(alkylene or substituted alkylene)-, —S(O)_(k)—where k is 1, 2, or 3, —S(O)_(k)(alkylene or substituted alkylene)-,—C(O)—, —NS(O)₂—, —OS(O)₂—, —C(O)-(alkylene or substituted alkylene)-,—C(S)—, —C(S)-(alkylene or substituted alkylene)-, —N(R′)—,—NR′-(alkylene or substituted alkylene)-, —C(O)N(R′)—,—CON(R′)-(alkylene or substituted alkylene)-, —CSN(R′)—,—CSN(R′)-(alkylene or substituted alkylene)-, —N(R′)CO-(alkylene orsubstituted alkylene)-, —N(R′)C(O)O—, —S(O)_(k)N(R′)—, —N(R′)C(O)N(R′)—,—N(R′)C(S)N(R′)—, —N(R′)S(O)_(k)N(R′)—, —N(R′)—N═, —C(R′)═N—,—C(R′)═N—N(R′)—, —C(R′)═N—N═, —C(R′)₂—N═N—, and —C(R′)₂—N(R′)—N(R′)—,where each R′ is independently H, alkyl, or substituted alkyl; R is H,alkyl, substituted alkyl, cycloalkyl, or substituted cycloalkyl; R₁ isH, an amino protecting group, resin, amino acid, polypeptide, orpolynucleotide; and R₂ is OH, an ester protecting group, resin, aminoacid, polypeptide, or polynucleotide; each R_(a) is independentlyselected from the group consisting of H, halogen, alkyl, substitutedalkyl, —N(R′)₂, —C(O)_(k)R′ where k is 1, 2, or 3, —C(O)N(R′)₂, —OR′,and —S(O)_(k)R′, where each R′ is independently H, alkyl, or substitutedalkyl; and n is 0 to 8. Such non-natural amino acids may be in the formof a salt, or may be incorporated into a non-natural amino acidpolypeptide, polymer, polysaccharide, or a polynucleotide and optionallypost translationally modified.

In addition, the following amino acids are included:

wherein such compounds are optionally amino protected, optionallycarboxyl protected, optionally amino protected and carboxyl protected,or a salt thereof, or may be incorporated into a non-natural amino acidpolypeptide, polymer, polysaccharide, or a polynucleotide and optionallypost translationally modified.

In addition to monocarbonyl structures, the non-natural amino acidsdescribed herein may include groups such as dicarbonyl, dicarbonyl like,masked dicarbonyl and protected dicarbonyl groups. For example, thefollowing amino acids having the structure of Formula (V) are included:

wherein, A is optional, and when present is lower alkylene, substitutedlower alkylene, lower cycloalkylene, substituted lower cycloalkylene,lower alkenylene, substituted lower alkenylene, alkynylene, lowerheteroalkylene, substituted heteroalkylene, lower heterocycloalkylene,substituted lower heterocycloalkylene, arylene, substituted arylene,heteroarylene, substituted heteroarylene, alkarylene, substitutedalkarylene, aralkylene, or substituted aralkylene; B is optional, andwhen present is a linker selected from the group consisting of loweralkylene, substituted lower alkylene, lower alkenylene, substitutedlower alkenylene, lower heteroalkylene, substituted lowerheteroalkylene, —O—, —O-(alkylene or substituted alkylene)-, —S—,—S-(alkylene or substituted alkylene)-, —S(O)_(k)— where k is 1, 2, or3, —S(O)_(k)(alkylene or substituted alkylene)-, —C(O)—, —NS(O)₂—,—OS(O)₂—, —C(O)-(alkylene or substituted alkylene)-, —C(S)—,—C(S)-(alkylene or substituted alkylene)-, —N(R′)—, —NR′-(alkylene orsubstituted alkylene)-, —C(O)N(R′)—, —CON(R′)-(alkylene or substitutedalkylene)-, —CSN(R′)—, —CSN(R′)-(alkylene or substituted alkylene)-,—N(R′)CO-(alkylene or substituted alkylene)-, —N(R′)C(O)O—,—S(O)_(k)N(R′)—, —N(R′)C(O)N(R′)—, —N(R′)C(S)N(R′)—,—N(R′)S(O)_(k)N(R′)—, —N(R′)—N═, —C(R′)═N—, —C(R′)═N—N(R′)—,—C(R′)═N—N═, —C(R′)₂—N═N—, and —C(R′)₂—N(R′)—N(R′)—, where each R′ isindependently H, alkyl, or substituted alkyl; R is H, alkyl, substitutedalkyl, cycloalkyl, or substituted cycloalkyl; R₁ is H, an aminoprotecting group, resin, amino acid, polypeptide, or polynucleotide; andR₂ is OH, an ester protecting group, resin, amino acid, polypeptide, orpolynucleotide. Such non-natural amino acids may be in the form of asalt, or may be incorporated into a non-natural amino acid polypeptide,polymer, polysaccharide, or a polynucleotide and optionally posttranslationally modified.

In addition, the following amino acids having the structure of Formula(VI) are included:

wherein, B is optional, and when present is a linker selected from thegroup consisting of lower alkylene, substituted lower alkylene, loweralkenylene, substituted lower alkenylene, lower heteroalkylene,substituted lower heteroalkylene, —O—, —O-(alkylene or substitutedalkylene)-, —S—, —S-(alkylene or substituted alkylene)-, —S(O)_(k)—where k is 1, 2, or 3, —S(O)_(k)(alkylene or substituted alkylene)-,—C(O)—, —NS(O)₂—, —OS(O)₂—, —C(O)-(alkylene or substituted alkylene)-,—C(S)—, —C(S)-(alkylene or substituted alkylene)-, —N(R′)—,—NR′-(alkylene or substituted alkylene)-, —C(O)N(R′)—,—CON(R′)-(alkylene or substituted alkylene)-, —CSN(R′)—,—CSN(R′)-(alkylene or substituted alkylene)-, —N(R′)CO-(alkylene orsubstituted alkylene)-, —N(R′)C(O)O—, —S(O)_(k)N(R′)—, —N(R′)C(O)N(R′)—,—N(R′)C(S)N(R′)—, —N(R′)S(O)_(k)N(R′)—, —N(R′)—N═, —C(R′)═N—,—C(R′)═N—N(R′)—, —C(R′)═N—N═, —C(R′)₂—N═N—, and —C(R′)₂—N(R′)—N(R′)—,where each R′ is independently H, alkyl, or substituted alkyl; R is H,alkyl, substituted alkyl, cycloalkyl, or substituted cycloalkyl; R₁ isH, an amino protecting group, resin, amino acid, polypeptide, orpolynucleotide; and R₂ is OH, an ester protecting group, resin, aminoacid, polypeptide, or polynucleotide; wherein each R_(a) isindependently selected from the group consisting of H, halogen, alkyl,substituted alkyl, —N(R′)₂, —C(O)_(k)R′ where k is 1, 2, or 3,—C(O)N(R′)₂, —OR′, and —S(O)_(k)R′, where each R′ is independently H,alkyl, or substituted alkyl. Such non-natural amino acids may be in theform of a salt, or may be incorporated into a non-natural amino acidpolypeptide, polymer, polysaccharide, or a polynucleotide and optionallypost translationally modified.

In addition, the following amino acids are included:

wherein such compounds are optionally amino protected and carboxylprotected, or a salt thereof. Such non-natural amino acids may be in theform of a salt, or may be incorporated into a non-natural amino acidpolypeptide, polymer, polysaccharide, or a polynucleotide and optionallypost translationally modified.

In addition, the following amino acids having the structure of Formula(VII) are included:

wherein, B is optional, and when present is a linker selected from thegroup consisting of lower alkylene, substituted lower alkylene, loweralkenylene, substituted lower alkenylene, lower heteroalkylene,substituted lower heteroalkylene, —O—, —O-(alkylene or substitutedalkylene)-, —S—, —S-(alkylene or substituted alkylene)-, —S(O)_(k)—where k is 1, 2, or 3, —S(O)_(k)(alkylene or substituted alkylene)-,—C(O)—, —NS(O)₂—, —OS(O)₂—, —C(O)-(alkylene or substituted alkylene)-,—C(S)—, —C(S)-(alkylene or substituted alkylene)-, —N(R′)—,—NR′-(alkylene or substituted alkylene)-, —C(O)N(R′)—,—CON(R′)-(alkylene or substituted alkylene)-, —CSN(R′)—,—CSN(R′)-(alkylene or substituted alkylene)-, —N(R′)CO-(alkylene orsubstituted alkylene)-, —N(R′)C(O)O—, —S(O)_(k)N(R′)—, —N(R′)C(O)N(R′)—,—N(R′)C(S)N(R′)—, —N(R′)S(O)_(k)N(R′)—, —N(R′)—N═, —C(R′)═N—,—C(R′)═N—N(R′)—, —C(R′)═N—N═, —C(R′)₂—N═N—, and —C(R′)₂—N(R′)—N(R′)—,where each R′ is independently H, alkyl, or substituted alkyl; R is H,alkyl, substituted alkyl, cycloalkyl, or substituted cycloalkyl; R₁ isH, an amino protecting group, resin, amino acid, polypeptide, orpolynucleotide; and R₂ is OH, an ester protecting group, resin, aminoacid, polypeptide, or polynucleotide; each R_(a) is independentlyselected from the group consisting of H, halogen, alkyl, substitutedalkyl, —N(R′)₂, —C(O)_(k)R′ where k is 1, 2, or 3, —C(O)N(R′)₂, —OR′,and —S(O)_(k)R′, where each R′ is independently H, alkyl, or substitutedalkyl; and n is 0 to 8. Such non-natural amino acids may be in the formof a salt, or may be incorporated into a non-natural amino acidpolypeptide, polymer, polysaccharide, or a polynucleotide and optionallypost translationally modified.

In addition, the following amino acids are included:

wherein such compounds are optionally amino protected and carboxylprotected, or a salt thereof, or may be incorporated into a non-naturalamino acid polypeptide, polymer, polysaccharide, or a polynucleotide andoptionally post translationally modified.

In addition, the following amino acids having the structure of Formula(XXX) are included:

wherein: A is optional, and when present is lower alkylene, substitutedlower alkylene, lower cycloalkylene, substituted lower cycloalkylene,lower alkenylene, substituted lower alkenylene, alkynylene, lowerheteroalkylene, substituted heteroalkylene, lower heterocycloalkylene,substituted lower heterocycloalkylene, arylene, substituted arylene,heteroarylene, substituted heteroarylene, alkarylene, substitutedalkarylene, aralkylene, or substituted aralkylene; R is H, alkyl,substituted alkyl, cycloalkyl, or substituted cycloalkyl; R₁ is H, anamino protecting group, resin, amino acid, polypeptide, orpolynucleotide; and R₂ is OH, an ester protecting group, resin, aminoacid, polypeptide, or polynucleotide; X₁ is C, S, or S(O); and L isalkylene, substituted alkylene, N(R′)(alkylene) or N(R′)(substitutedalkylene), where R′ is H, alkyl, substituted alkyl, cycloalkyl, orsubstituted cycloalkyl. Such non-natural amino acids may be in the formof a salt, or may be incorporated into a non-natural amino acidpolypeptide, polymer, polysaccharide, or a polynucleotide and optionallypost translationally modified.

In addition, the following amino acids having the structure of Formula(XXX-A) are included:

wherein: A is optional, and when present is lower alkylene, substitutedlower alkylene, lower cycloalkylene, substituted lower cycloalkylene,lower alkenylene, substituted lower alkenylene, alkynylene, lowerheteroalkylene, substituted heteroalkylene, lower heterocycloalkylene,substituted lower heterocycloalkylene, arylene, substituted arylene,heteroarylene, substituted heteroarylene, alkarylene, substitutedalkarylene, aralkylene, or substituted aralkylene; R is H, alkyl,substituted alkyl, cycloalkyl, or substituted cycloalkyl; R₁ is H, anamino protecting group, resin, amino acid, polypeptide, orpolynucleotide; and R₂ is OH, an ester protecting group, resin, aminoacid, polypeptide, or polynucleotide; L is alkylene, substitutedalkylene, N(R′)(alkylene) or N(R′)(substituted alkylene), where R′ is H,alkyl, substituted alkyl, cycloalkyl, or substituted cycloalkyl. Suchnon-natural amino acids may be in the form of a salt, or may beincorporated into a non-natural amino acid polypeptide, polymer,polysaccharide, or a polynucleotide and optionally post translationallymodified.

In addition, the following amino acids having the structure of Formula(XXX-B) are included:

wherein: A is optional, and when present is lower alkylene, substitutedlower alkylene, lower cycloalkylene, substituted lower cycloalkylene,lower alkenylene, substituted lower alkenylene, alkynylene, lowerheteroalkylene, substituted heteroalkylene, lower heterocycloalkylene,substituted lower heterocycloalkylene, arylene, substituted arylene,heteroarylene, substituted heteroarylene, alkarylene, substitutedalkarylene, aralkylene, or substituted aralkylene; R is H, alkyl,substituted alkyl, cycloalkyl, or substituted cycloalkyl; R₁ is H, anamino protecting group, resin, amino acid, polypeptide, orpolynucleotide; and R₂ is OH, an ester protecting group, resin, aminoacid, polypeptide, or polynucleotide; L is alkylene, substitutedalkylene, N(R′)(alkylene) or N(R′)(substituted alkylene), where R′ is H,alkyl, substituted alkyl, cycloalkyl, or substituted cycloalkyl. Suchnon-natural amino acids may be in the form of a salt, or may beincorporated into a non-natural amino acid polypeptide, polymer,polysaccharide, or a polynucleotide and optionally post translationallymodified.

In addition, the following amino acids having the structure of Formula(XXXI) are included:

wherein: A is optional, and when present is lower alkylene, substitutedlower alkylene, lower cycloalkylene, substituted lower cycloalkylene,lower alkenylene, substituted lower alkenylene, alkynylene, lowerheteroalkylene, substituted heteroalkylene, lower heterocycloalkylene,substituted lower heterocycloalkylene, arylene, substituted arylene,heteroarylene, substituted heteroarylene, alkarylene, substitutedalkarylene, aralkylene, or substituted aralkylene; R is H, alkyl,substituted alkyl, cycloalkyl, or substituted cycloalkyl; R₁ is H, anamino protecting group, resin, amino acid, polypeptide, orpolynucleotide; and R₂ is OH, an ester protecting group, resin, aminoacid, polypeptide, or polynucleotide; X₁ is C, S, or S(O); and n is 0,1, 2, 3, 4, or 5; and each R⁸ and R⁹ on each CR⁸R⁹ group isindependently selected from the group consisting of H, alkoxy,alkylamine, halogen, alkyl, aryl, or any R⁸ and R⁹ can together form ═Oor a cycloalkyl, or any to adjacent R⁸ groups can together form acycloalkyl. Such non-natural amino acids may be in the form of a salt,or may be incorporated into a non-natural amino acid polypeptide,polymer, polysaccharide, or a polynucleotide and optionally posttranslationally modified.

In addition, the following amino acids having the structure of Formula(XXXI-A) are included:

wherein: A is optional, and when present is lower alkylene, substitutedlower alkylene, lower cycloalkylene, substituted lower cycloalkylene,lower alkenylene, substituted lower alkenylene, alkynylene, lowerheteroalkylene, substituted heteroalkylene, lower heterocycloalkylene,substituted lower heterocycloalkylene, arylene, substituted arylene,heteroarylene, substituted heteroarylene, alkarylene, substitutedalkarylene, aralkylene, or substituted aralkylene; R is H, alkyl,substituted alkyl, cycloalkyl, or substituted cycloalkyl; R₁ is H, anamino protecting group, resin, amino acid, polypeptide, orpolynucleotide; and R₂ is OH, an ester protecting group, resin, aminoacid, polypeptide, or polynucleotide; n is 0, 1, 2, 3, 4, or 5; and eachR⁸ and R⁹ on each CR⁸R⁹ group is independently selected from the groupconsisting of H, alkoxy, alkylamine, halogen, alkyl, aryl, or any R⁸ andR⁹ can together form ═O or a cycloalkyl, or any to adjacent R⁸ groupscan together form a cycloalkyl. Such non-natural amino acids may be inthe form of a salt, or may be incorporated into a non-natural amino acidpolypeptide, polymer, polysaccharide, or a polynucleotide and optionallypost translationally modified.

In addition, the following amino acids having the structure of Formula(XXXI-B) are included:

wherein: A is optional, and when present is lower alkylene, substitutedlower alkylene, lower cycloalkylene, substituted lower cycloalkylene,lower alkenylene, substituted lower alkenylene, alkynylene, lowerheteroalkylene, substituted heteroalkylene, lower heterocycloalkylene,substituted lower heterocycloalkylene, arylene, substituted arylene,heteroarylene, substituted heteroarylene, alkarylene, substitutedalkarylene, aralkylene, or substituted aralkylene; R is H, alkyl,substituted alkyl, cycloalkyl, or substituted cycloalkyl; R₁ is H, anamino protecting group, resin, amino acid, polypeptide, orpolynucleotide; and R₂ is OH, an ester protecting group, resin, aminoacid, polypeptide, or polynucleotide; n is 0, 1, 2, 3, 4, or 5; and eachR⁸ and R⁹ on each CR⁸R⁹ group is independently selected from the groupconsisting of H, alkoxy, alkylamine, halogen, alkyl, aryl, or any R⁸ andR⁹ can together form ═O or a cycloalkyl, or any to adjacent R⁸ groupscan together form a cycloalkyl. Such non-natural amino acids may be inthe form of a salt, or may be incorporated into a non-natural amino acidpolypeptide, polymer, polysaccharide, or a polynucleotide and optionallypost translationally modified.

In addition, the following amino acids having the structure of Formula(XXXII) are included:

wherein: A is optional, and when present is lower alkylene, substitutedlower alkylene, lower cycloalkylene, substituted lower cycloalkylene,lower alkenylene, substituted lower alkenylene, alkynylene, lowerheteroalkylene, substituted heteroalkylene, lower heterocycloalkylene,substituted lower heterocycloalkylene, arylene, substituted arylene,heteroarylene, substituted heteroarylene, alkarylene, substitutedalkarylene, aralkylene, or substituted aralkylene; R is H, alkyl,substituted alkyl, cycloalkyl, or substituted cycloalkyl; R₁ is H, anamino protecting group, resin, amino acid, polypeptide, orpolynucleotide; and R₂ is OH, an ester protecting group, resin, aminoacid, polypeptide, or polynucleotide; X₁ is C, S, or S(O); and L isalkylene, substituted alkylene, N(R′)(alkylene) or N(R′)(substitutedalkylene), where R′ is H, alkyl, substituted alkyl, cycloalkyl, orsubstituted cycloalkyl. Such non-natural amino acids may be in the formof a salt, or may be incorporated into a non-natural amino acidpolypeptide, polymer, polysaccharide, or a polynucleotide and optionallypost translationally modified.

The In addition, the following amino acids having the structure ofFormula (XXXII-A) are included:

wherein: A is optional, and when present is lower alkylene, substitutedlower alkylene, lower cycloalkylene, substituted lower cycloalkylene,lower alkenylene, substituted lower alkenylene, alkynylene, lowerheteroalkylene, substituted heteroalkylene, lower heterocycloalkylene,substituted lower heterocycloalkylene, arylene, substituted arylene,heteroarylene, substituted heteroarylene, alkarylene, substitutedalkarylene, aralkylene, or substituted aralkylene; R is H, alkyl,substituted alkyl, cycloalkyl, or substituted cycloalkyl; R₁ is H, anamino protecting group, resin, amino acid, polypeptide, orpolynucleotide; and R₂ is OH, an ester protecting group, resin, aminoacid, polypeptide, or polynucleotide; L is alkylene, substitutedalkylene, N(R′)(alkylene) or N(R′)(substituted alkylene), where R′ is H,alkyl, substituted alkyl, cycloalkyl, or substituted cycloalkyl. Suchnon-natural amino acids may be in the form of a salt, or may beincorporated into a non-natural amino acid polypeptide, polymer,polysaccharide, or a polynucleotide and optionally post translationallymodified.

In addition, the following amino acids having the structure of Formula(XXXII-B) are included:

wherein: A is optional, and when present is lower alkylene, substitutedlower alkylene, lower cycloalkylene, substituted lower cycloalkylene,lower alkenylene, substituted lower alkenylene, alkynylene, lowerheteroalkylene, substituted heteroalkylene, lower heterocycloalkylene,substituted lower heterocycloalkylene, arylene, substituted arylene,heteroarylene, substituted heteroarylene, alkarylene, substitutedalkarylene, aralkylene, or substituted aralkylene; R is H, alkyl,substituted alkyl, cycloalkyl, or substituted cycloalkyl; R₁ is H, anamino protecting group, resin, amino acid, polypeptide, orpolynucleotide; and R₂ is OH, an ester protecting group, resin, aminoacid, polypeptide, or polynucleotide; L is alkylene, substitutedalkylene, N(R′)(alkylene) or N(R′)(substituted alkylene), where R′ is H,alkyl, substituted alkyl, cycloalkyl, or substituted cycloalkyl. Suchnon-natural amino acids may be in the form of a salt, or may beincorporated into a non-natural amino acid polypeptide, polymer,polysaccharide, or a polynucleotide and optionally post translationallymodified.

In addition, amino acids having the structure of Formula (XXXX) areincluded:

wherein: A is optional, and when present is lower alkylene, substitutedlower alkylene, lower cycloalkylene, substituted lower cycloalkylene,lower alkenylene, substituted lower alkenylene, alkynylene, lowerheteroalkylene, substituted heteroalkylene, lower heterocycloalkylene,substituted lower heterocycloalkylene, arylene, substituted arylene,heteroarylene, substituted heteroarylene, alkarylene, substitutedalkarylene, aralkylene, or substituted aralkylene;

where (a) indicates bonding to the A group and (b) indicates bonding torespective carbonyl groups, R₃ and R₄ are independently chosen from H,halogen, alkyl, substituted alkyl, cycloalkyl, or substitutedcycloalkyl, or R₃ and R₄ or two R₃ groups or two R₄ groups optionallyform a cycloalkyl or a heterocycloalkyl; R is H, halogen, alkyl,substituted alkyl, cycloalkyl, or substituted cycloalkyl; T₃ is a bond,C(R)(R), O, or S, and R is H, halogen, alkyl, substituted alkyl,cycloalkyl, or substituted cycloalkyl; R₁ is H, an amino protectinggroup, resin, amino acid, polypeptide, or polynucleotide; and R₂ is OH,an ester protecting group, resin, amino acid, polypeptide, orpolynucleotide. Such non-natural amino acids may be in the form of asalt, or may be incorporated into a non-natural amino acid polypeptide,polymer, polysaccharide, or a polynucleotide and optionally posttranslationally modified.

In addition, amino acids having the structure of Formula (XXXXI) areincluded:

wherein:

where (a) indicates bonding to the A group and (b) indicates bonding torespective carbonyl groups, R₃ and R₄ are independently chosen from H,halogen, alkyl, substituted alkyl, cycloalkyl, or substitutedcycloalkyl, or R₃ and R₄ or two R₃ groups or two R₄ groups optionallyform a cycloalkyl or a heterocycloalkyl; R is H, halogen, alkyl,substituted alkyl, cycloalkyl, or substituted cycloalkyl; T₃ is a bond,C(R)(R), O, or S, and R is H, halogen, alkyl, substituted alkyl,cycloalkyl, or substituted cycloalkyl; R₁ is H, an amino protectinggroup, resin, amino acid, polypeptide, or polynucleotide; and R₂ is OH,an ester protecting group, resin, amino acid, polypeptide, orpolynucleotide; each R_(a) is independently selected from the groupconsisting of H, halogen, alkyl, substituted alkyl, —N(R′)₂, —C(O)_(k)R′where k is 1, 2, or 3, —C(O)N(R′)₂, —OR′, and —S(O)_(k)R′, where each R′is independently H, alkyl, or substituted alkyl. Such non-natural aminoacids may be in the form of a salt, or may be incorporated into anon-natural amino acid polypeptide, polymer, polysaccharide, or apolynucleotide and optionally post translationally modified.

In addition, amino acids having the structure of Formula (XXXXII) areincluded:

wherein: R is H, halogen, alkyl, substituted alkyl, cycloalkyl, orsubstituted cycloalkyl; and T₃ is O, or S. Such non-natural amino acidsmay be in the form of a salt, or may be incorporated into a non-naturalamino acid polypeptide, polymer, polysaccharide, or a polynucleotide andoptionally post translationally modified.

In addition, amino acids having the structure of Formula (XXXXIII) areincluded:

wherein: R is H, halogen, alkyl, substituted alkyl, cycloalkyl, orsubstituted cycloalkyl.

In addition, the following amino acids having structures of Formula(XXXXIII) are included:

Such non-natural amino acids may be in the form of a salt, or may beincorporated into a non-natural amino acid polypeptide, polymer,polysaccharide, or a polynucleotide and optionally post translationallymodified.

Non-natural amino acids containing a hydroxylamine (also called anaminooxy) group allow for reaction with a variety of electrophilicgroups to form conjugates (including but not limited to, with PEG orother water soluble polymers). Like hydrazines, hydrazides andsemicarbazides, the enhanced nucleophilicity of the aminooxy grouppermits it to react efficiently and selectively with a variety ofmolecules that contain carbonyl- or dicarbonyl-groups, including but notlimited to, ketones, aldehydes or other functional groups with similarchemical reactivity. See, e.g., Shao, J. and Tam, J., J. Am. Chem. Soc.117:3893-3899 (1995); H. Hang and C. Bertozzi, Acc. Chem. Res. 34(9):727-736 (2001). Whereas the result of reaction with a hydrazine group isthe corresponding hydrazone, however, an oxime results generally fromthe reaction of an aminooxy group with a carbonyl- ordicarbonyl-containing group such as, by way of example, a ketones,aldehydes or other functional groups with similar chemical reactivity.

Thus, in certain embodiments described herein are non-natural aminoacids with sidechains comprising a hydroxylamine group, ahydroxylamine-like group (which has reactivity similar to ahydroxylamine group and is structurally similar to a hydroxylaminegroup), a masked hydroxylamine group (which can be readily convertedinto a hydroxylamine group), or a protected hydroxylamine group (whichhas reactivity similar to a hydroxylamine group upon deprotection). Suchamino acids include amino acids having the structure of Formula (XIV):

wherein: A is optional, and when present is lower alkylene, substitutedlower alkylene, lower cycloalkylene, substituted lower cycloalkylene,lower alkenylene, substituted lower alkenylene, alkynylene, lowerheteroalkylene, substituted heteroalkylene, lower heterocycloalkylene,substituted lower heterocycloalkylene, arylene, substituted arylene,heteroarylene, substituted heteroarylene, alkarylene, substitutedalkarylene, aralkylene, or substituted aralkylene; B is optional, andwhen present is a linker selected from the group consisting of loweralkylene, substituted lower alkylene, lower alkenylene, substitutedlower alkenylene, lower heteroalkylene, substituted lowerheteroalkylene, —O—, —O-(alkylene or substituted alkylene)-, —S—,—S-(alkylene or substituted alkylene)-, —S(O)_(k)— where k is 1, 2, or3, —S(O)_(k)(alkylene or substituted alkylene)-, —C(O)—, —NS(O)₂—,—OS(O)₂—, —C(O)-(alkylene or substituted alkylene)-, —C(S)—,—C(S)-(alkylene or substituted alkylene)-, —N(R′)—, —NR′-(alkylene orsubstituted alkylene)-, —C(O)N(R′)—, —CON(R′)-(alkylene or substitutedalkylene)-, —CSN(R′)—, —CSN(R′)-(alkylene or substituted alkylene)-,—N(R′)CO-(alkylene or substituted alkylene)-, —N(R′)C(O)O—,—S(O)_(k)N(R′)—, —N(R′)C(O)N(R′)—, —N(R′)C(S)N(R′)—,—N(R′)S(O)_(k)N(R′)—, —N(R′)—N═, —C(R′)═N—, —C(R′)═N—N(R′)—,—C(R′)═N—N═, —C(R′)₂—N═N—, and —C(R′)₂—N(R′)—N(R′)—, where each R′ isindependently H, alkyl, or substituted alkyl; K is —NR₆R₇ or —N═CR₆R₇;R₁ is H, an amino protecting group, resin, amino acid, polypeptide, orpolynucleotide; and R₂ is OH, an ester protecting group, resin, aminoacid, polypeptide, or polynucleotide; each of R₃ and R₄ is independentlyH, halogen, lower alkyl, or substituted lower alkyl, or R₃ and R₄ or twoR₃ groups optionally form a cycloalkyl or a heterocycloalkyl; each of R₆and R₇ is independently selected from the group consisting of H, alkyl,substituted alkyl, alkenyl, substituted alkenyl, alkoxy, substitutedalkoxy, polyalkylene oxide, substituted polyalkylene oxide, aryl,substituted aryl, heteroaryl, substituted heteroaryl, alkaryl,substituted alkaryl, aralkyl, and substituted aralkyl, —C(O)R″,—C(O)₂R″, —C(O)N(R″)₂, wherein each R″ is independently hydrogen, alkyl,substituted alkyl, alkenyl, substituted alkenyl, alkoxy, substitutedalkoxy, aryl, substituted aryl, heteroaryl, alkaryl, substitutedalkaryl, aralkyl, or substituted aralkyl; or R₆ or R₇ is L-X, where X isa selected from the group consisting of a label; a dye; a polymer; awater-soluble polymer; a derivative of polyethylene glycol; aphotocrosslinker; a cytotoxic compound; a drug; an affinity label; aphotoaffinity label; a reactive compound; a resin; a second protein orpolypeptide or polypeptide analog; an antibody or antibody fragment; ametal chelator; a cofactor; a fatty acid; a carbohydrate; apolynucleotide; a DNA; a RNA; an antisense polynucleotide; a saccharide,a water-soluble dendrimer, a cyclodextrin, a biomaterial; ananoparticle; a spin label; a fluorophore, a metal-containing moiety; aradioactive moiety; a novel functional group; a group that covalently ornoncovalently interacts with other molecules; a photocaged moiety; aphotoisomerizable moiety; biotin; a biotin analogue; a moietyincorporating a heavy atom; a chemically cleavable group; aphotocleavable group; an elongated side chain; a carbon-linked sugar; aredox-active agent; an amino thioacid; a toxic moiety; an isotopicallylabeled moiety; a biophysical probe; a phosphorescent group; achemiluminescent group; an electron dense group; a magnetic group; anintercalating group; a chromophore; an energy transfer agent; abiologically active agent; a detectable label; and any combinationthereof; and L is optional, and when present is a linker selected fromthe group consisting of alkylene, substituted alkylene, alkenylene,substituted alkenylene, —O—, —O-(alkylene or substituted alkylene)-,—S—, —S-(alkylene or substituted alkylene)-, —S(O)_(k)— where k is 1, 2,or 3, —S(O)_(k)(alkylene or substituted alkylene)-, —C(O)—,—C(O)-(alkylene or substituted alkylene)-, —C(S)—, —C(S)-(alkylene orsubstituted alkylene)-, —N(R′)—, —NR′-(alkylene or substitutedalkylene)-, —C(O)N(R′)—, —CON(R′)-(alkylene or substituted alkylene)-,—CSN(R′)—, —CSN(R′)-(alkylene or substituted alkylene)-,—N(R′)CO-(alkylene or substituted alkylene)-, —N(R′)C(O)O—,—S(O)_(k)N(R′)—, —N(R′)C(O)N(R′)—, —N(R′)C(S)N(R′)—,—N(R′)S(O)_(k)N(R′)—, —N(R′)—N═, —C(R′)═N—, —C(R′)═N—N(R′)—,—C(R′)═N—N═, —C(R′)₂—N═N—, and —C(R′)₂—N(R′)—N(R′)—, where each R′ isindependently H, alkyl, or substituted alkyl. Such non-natural aminoacids may be in the form of a salt, or may be incorporated into anon-natural amino acid polypeptide, polymer, polysaccharide, or apolynucleotide and optionally post translationally modified.

In certain embodiments of compounds of Formula (XIV), A is phenylene orsubstituted phenylene. In certain embodiments of compounds of Formula(XIV), B is -(alkylene or substituted alkylene)-, —O-(alkylene orsubstituted alkylene)-, —S-(alkylene or substituted alkylene)-, or—C(O)-(alkylene or substituted alkylene)-. In certain embodiments ofcompounds of Formula (XIV), B is —O(CH₂)₂—, —S(CH₂)₂—, —NH(CH₂)₂—,—CO(CH₂)₂—, or —(CH₂)— where n is 1 to 4. In certain embodiments ofcompounds of Formula (XIV), R₁ is H, tert-butyloxycarbonyl (Boc),9-Fluorenylmethoxycarbonyl (Fmoc), N-acetyl, tetrafluoroacetyl (TFA), orbenzyloxycarbonyl (Cbz). In certain embodiments of compounds of Formula(XIV), R₁ is a resin, amino acid, polypeptide, or polynucleotide. Incertain embodiments of compounds of Formula (XIV), wherein R₂ is OH,O-methyl, O-ethyl, or O-t-butyl. In certain embodiments of compounds ofFormula (XIV), R₂ is a resin, amino acid, polypeptide, orpolynucleotide. In certain embodiments of compounds of Formula (XIV), R₂is a polynucleotide. In certain embodiments of compounds of Formula(XIV), R₂ is ribonucleic acid (RNA). In certain embodiments of compoundsof Formula (XIV), R₂ is tRNA. In certain embodiments of compounds ofFormula (XIV), the tRNA specifically recognizes a codon selected fromthe group consisting of an amber codon, ochre codon, opal codon, aunique codon, a rare codon, an unnatural codon, a five-base codon, and afour-base codon. In certain embodiments of compounds of Formula (XIV),R₂ is a suppressor tRNA. In certain embodiments of compounds of Formula(XIV), each of R₆ and R₇ is independently selected from the groupconsisting of H, alkyl, substituted alkyl, alkoxy, substituted alkoxy,polyalkylene oxide, substituted polyalkylene oxide, aryl, substitutedaryl, heteroaryl, substituted heteroaryl, alkaryl, substituted alkaryl,aralkyl, and substituted aralkyl. In certain embodiments of compounds ofFormula (XIV), each of R₆ and R₇ is independently selected from thegroup consisting of H, methyl, phenyl, and -[(alkylene or substitutedalkylene)-O-(hydrogen, alkyl, or substituted alkyl)]_(x), wherein x isfrom 1-50. In certain embodiments of compounds of Formula (XIV), K is—NR₆R₇.

In certain embodiments of compounds of Formula (XIV), X is abiologically active agent selected from the group consisting of apeptide, protein, enzyme, antibody, drug, dye, lipid, nucleosides,oligonucleotide, cell, virus, liposome, microparticle, and micelle. Incertain embodiments of compounds of Formula (XIV), X is a drug selectedfrom the group consisting of an antibiotic, fungicide, anti-viral agent,anti-inflammatory agent, anti-tumor agent, cardiovascular agent,anti-anxiety agent, hormone, growth factor, and steroidal agent. Incertain embodiments of compounds of Formula (XIV), X is an enzymeselected from the group consisting of horseradish peroxidase, alkalinephosphatase, β-galactosidase, and glucose oxidase. In certainembodiments of compounds of Formula (XIV), X is a detectable labelselected from the group consisting of a fluorescent, phosphorescent,chemiluminescent, chelating, electron dense, magnetic, intercalating,radioactive, chromophoric, and energy transfer moiety.

In certain embodiments, compounds of Formula (XIV) are stable in aqueoussolution for at least 1 month under mildly acidic conditions. In certainembodiments, compounds of Formula (XIV) are stable for at least 2 weeksunder mildly acidic conditions. In certain embodiments, compound ofFormula (XIV) are stable for at least 5 days under mildly acidicconditions. In certain embodiments, such acidic conditions are pH 2 to8.

Such amino acids include amino acids having the structure of Formula(XV):

wherein A is optional, and when present is lower alkylene, substitutedlower alkylene, lower cycloalkylene, substituted lower cycloalkylene,lower alkenylene, substituted lower alkenylene, alkynylene, lowerheteroalkylene, substituted heteroalkylene, lower heterocycloalkylene,substituted lower heterocycloalkylene, arylene, substituted arylene,heteroarylene, substituted heteroarylene, alkarylene, substitutedalkarylene, aralkylene, or substituted aralkylene; B is optional, andwhen present is a linker selected from the group consisting of loweralkylene, substituted lower alkylene, lower alkenylene, substitutedlower alkenylene, lower heteroalkylene, substituted lowerheteroalkylene, —O—, —O-(alkylene or substituted alkylene)-, —S—,—S-(alkylene or substituted alkylene)-, —S(O)_(k)— where k is 1, 2, or3, —S(O)_(k)(alkylene or substituted alkylene)-, —C(O)—, —NS(O)₂—,—OS(O)₂—, —C(O)-(alkylene or substituted alkylene)-, —C(S)—,—C(S)-(alkylene or substituted alkylene)-, —N(R′)—, —NR′-(alkylene orsubstituted alkylene)-, —C(O)N(R′)—, —CON(R′)-(alkylene or substitutedalkylene)-, —CSN(R′)—, —CSN(R′)-(alkylene or substituted alkylene)-,—N(R′)CO-(alkylene or substituted alkylene)-, —N(R′)C(O)O—,—S(O)_(k)N(R′)—, —N(R′)C(O)N(R′)—, —N(R′)C(S)N(R′)—,—N(R′)S(O)_(k)N(R′)—, —N(R′)—N═, —C(R′)═N—, —C(R′)═N—N(R′)—,—C(R′)═N—N═, —C(R′)₂—N═N—, and —C(R′)₂—N(R′)—N(R′)—, where each R′ isindependently H, alkyl, or substituted alkyl; R₁ is H, an aminoprotecting group, resin, amino acid, polypeptide, or polynucleotide; andR₂ is OH, an ester protecting group, resin, amino acid, polypeptide, orpolynucleotide; each of R₃ and R₄ is independently H, halogen, loweralkyl, or substituted lower alkyl, or R₃ and R₄ or two R₃ groupsoptionally form a cycloalkyl or a heterocycloalkyl. Such non-naturalamino acids may be in the form of a salt, or may be incorporated into anon-natural amino acid polypeptide, polymer, polysaccharide, or apolynucleotide and optionally post translationally modified.

A non-limiting, representative amino acid has the following structure:

Such a non-natural amino acid may be in the form of a salt, or may beincorporated into a non-natural amino acid polypeptide, polymer,polysaccharide, or a polynucleotide and optionally post translationallymodified.

Non-natural amino acids containing an oxime group allow for reactionwith a variety of reagents that contain certain reactive carbonyl- ordicarbonyl-groups (including but not limited to, ketones, aldehydes, orother groups with similar reactivity) to form new non-natural aminoacids comprising a new oxime group. Such an oxime exchange reactionallow for the further functionalization of non-natural amino acidpolypeptides. Further, the original non-natural amino acids containingan oxime group may be useful in their own right as long as the oximelinkage is stable under conditions necessary to incorporate the aminoacid into a polypeptide (e.g., the in vivo, in vitro and chemicalsynthetic methods described herein).

Thus, in certain embodiments described herein are non-natural aminoacids with sidechains comprising an oxime group, an oxime-like group(which has reactivity similar to an oxime group and is structurallysimilar to an oxime group), a masked oxime group (which can be readilyconverted into an oxime group), or a protected oxime group (which hasreactivity similar to an oxime group upon deprotection). Such aminoacids include amino acids having the structure of Formula (XI):

wherein: A is optional, and when present is lower alkylene, substitutedlower alkylene, lower cycloalkylene, substituted lower cycloalkylene,lower alkenylene, substituted lower alkenylene, alkynylene, lowerheteroalkylene, substituted heteroalkylene, lower heterocycloalkylene,substituted lower heterocycloalkylene, arylene, substituted arylene,heteroarylene, substituted heteroarylene, alkarylene, substitutedalkarylene, aralkylene, or substituted aralkylene; B is optional, andwhen present is a linker selected from the group consisting of loweralkylene, substituted lower alkylene, lower alkenylene, substitutedlower alkenylene, lower heteroalkylene, substituted lowerheteroalkylene, —O—, —O-(alkylene or substituted alkylene)-, —S—,—S-(alkylene or substituted alkylene)-, —S(O)_(k)— where k is 1, 2, or3, —S(O)_(k)(alkylene or substituted alkylene)-, —C(O)—, —NS(O)₂—,—OS(O)₂—, —C(O)-(alkylene or substituted alkylene)-, —C(S)—,—C(S)-(alkylene or substituted alkylene)-, —N(R′)—, —NR′-(alkylene orsubstituted alkylene)-, —C(O)N(R′)—, —CON(R′)-(alkylene or substitutedalkylene)-, —CSN(R′)—, —CSN(R′)-(alkylene or substituted alkylene)-,—N(R′)CO-(alkylene or substituted alkylene)-, —N(R′)C(O)O—,—S(O)_(k)N(R′)—, —N(R′)C(O)N(R′)—, —N(R′)C(S)N(R′)—,—N(R′)S(O)_(k)N(R′)—, —N(R′)—N═, —C(R′)═N—, —C(R′)═N—N(R′)—,—C(R′)═N—N═, —C(R′)₂—N═N—, and —C(R′)₂—N(R′)—N(R′)—, where each R′ isindependently H, alkyl, or substituted alkyl; R is H, alkyl, substitutedalkyl, cycloalkyl, or substituted cycloalkyl; R₁ is H, an aminoprotecting group, resin, amino acid, polypeptide, or polynucleotide; andR₂ is OH, an ester protecting group, resin, amino acid, polypeptide, orpolynucleotide; each of R₃ and R₄ is independently H, halogen, loweralkyl, or substituted lower alkyl, or R₃ and R₄ or two R₃ optionallyform a cycloalkyl or a heterocycloalkyl; R₅ is H, alkyl, substitutedalkyl, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl,alkoxy, substituted alkoxy, alkylalkoxy, substituted alkylalkoxy,polyalkylene oxide, substituted polyalkylene oxide, aryl, substitutedaryl, heteroaryl, substituted heteroaryl, alkaryl, substituted alkaryl,aralkyl, substituted aralkyl, -(alkylene or substitutedalkylene)-ON(R″)₂, -(alkylene or substituted alkylene)-C(O)SR″,-(alkylene or substituted alkylene)-S—S-(aryl or substituted aryl),—C(O)R″, —C(O)₂R″, or —C(O)N(R″)₂, wherein each R″ is independentlyhydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl,alkoxy, substituted alkoxy, aryl, substituted aryl, heteroaryl, alkaryl,substituted alkaryl, aralkyl, or substituted aralkyl; or R₅ is L-X,where X is a selected from the group consisting of a label; a dye; apolymer; a water-soluble polymer; a derivative of polyethylene glycol; aphotocrosslinker; a cytotoxic compound; a drug; an affinity label; aphotoaffinity label; a reactive compound; a resin; a second protein orpolypeptide or polypeptide analog; an antibody or antibody fragment; ametal chelator; a cofactor; a fatty acid; a carbohydrate; apolynucleotide; a DNA; a RNA; an antisense polynucleotide; a saccharide,a water-soluble dendrimer, a cyclodextrin, a biomaterial; ananoparticle; a spin label; a fluorophore, a metal-containing moiety; aradioactive moiety; a novel functional group; a group that covalently ornoncovalently interacts with other molecules; a photocaged moiety; aphotoisomerizable moiety; biotin; a biotin analogue; a moietyincorporating a heavy atom; a chemically cleavable group; aphotocleavable group; an elongated side chain; a carbon-linked sugar; aredox-active agent; an amino thioacid; a toxic moiety; an isotopicallylabeled moiety; a biophysical probe; a phosphorescent group; achemiluminescent group; an electron dense group; a magnetic group; anintercalating group; a chromophore; an energy transfer agent; abiologically active agent; a detectable label; and any combinationthereof; and L is optional, and when present is a linker selected fromthe group consisting of alkylene, substituted alkylene, alkenylene,substituted alkenylene, —O—, —O-(alkylene or substituted alkylene)-,—S—, —S-(alkylene or substituted alkylene)-, —S(O)_(k)— where k is 1, 2,or 3, —S(O)_(k)(alkylene or substituted alkylene)-, —C(O)—,—C(O)-(alkylene or substituted alkylene)-, —C(S)—, —C(S)-(alkylene orsubstituted alkylene)-, —N(R′)—, —NR′-(alkylene or substitutedalkylene)-, —C(O)N(R′)—, —CON(R′)-(alkylene or substituted alkylene)-,—CSN(R′)—, —CSN(R′)-(alkylene or substituted alkylene)-,—N(R′)CO-(alkylene or substituted alkylene)-, —N(R′)C(O)O—, -(alkyleneor substituted alkylene)-O—N═CR′—, -(alkylene or substitutedalkylene)-C(O)NR′-(alkylene or substituted alkylene)-, -(alkylene orsubstituted alkylene)-S(O)_(k)-(alkylene or substituted alkylene)-S—,-(alkylene or substituted alkylene)-S—S—, —S(O)_(k)N(R′)—,—N(R′)C(O)N(R′)—, —N(R′)C(S)N(R′)—, —N(R′)S(O)_(k)N(R′)—, —N(R′)—N═,—C(R′)═N—, —C(R′)═N—N(R′)—, —C(R′)═N—N═, —C(R′)₂—N═N—, and—C(R′)₂—N(R′)—N(R′)—, where each R′ is independently H, alkyl, orsubstituted alkyl; with a proviso that when A and B are absent, R is notmethyl. Such non-natural amino acids may be in the form of a salt, ormay be incorporated into a non-natural amino acid polypeptide, polymer,polysaccharide, or a polynucleotide and optionally post translationallymodified.

In certain embodiments of compounds of Formula (XI), B is —O-(alkyleneor substituted alkylene)-. In certain embodiments of compounds ofFormula (XI), B is —O(CH₂)—. In certain embodiments of compounds ofFormula (XI), R is C₁₋₄ alkyl. In certain embodiments of compounds ofFormula (XI), R is —CH₃. In certain embodiments of compounds of Formula(XI), R₁ is H, tert-butyloxycarbonyl (Boc), 9-fluorenylmethoxycarbonyl(Fmoc), N-acetyl, tetrafluoroacetyl (TFA), or benzyloxycarbonyl (Cbz).In certain embodiments of compounds of Formula (XI), R₁ is a resin,amino acid, polypeptide, or polynucleotide. In certain embodiments ofcompounds of Formula (XI), R₂ is OH, O-methyl, O-ethyl, or O-t-butyl. Incertain embodiments of compounds of Formula (XI), R₂ is a resin, aminoacid, polypeptide, or polynucleotide. In certain embodiments ofcompounds of Formula (XI), R₂ is a polynucleotide. In certainembodiments of compounds of Formula (XI), R₂ is ribonucleic acid (RNA).In certain embodiments of compounds of Formula (XI), R₂ is tRNA. Incertain embodiments of compounds of Formula (XI), the tRNA specificallyrecognizes a selector codon. In certain embodiments of compounds ofFormula (XI), the selector codon is selected from the group consistingof an amber codon, ochre codon, opal codon, a unique codon, a rarecodon, an unnatural codon, a five-base codon, and a four-base codon. Incertain embodiments of compounds of Formula (XI), R₂ is a suppressortRNA. In certain embodiments of compounds of Formula (XI), R₅ isalkylalkoxy, substituted alkylalkoxy, polyalkylene oxide, substitutedpolyalkylene oxide, or —C(O)₂R″. In certain embodiments of compounds ofFormula (XI), R₅ is -[(alkylene or substituted alkylene)-O-(hydrogen,alkyl, or substituted alkyl)]_(x), wherein x is from 1-50. In certainembodiments of compounds of Formula (XI), R₅ is —(CH₂CH₂)—O—CH₃ or—COOH.

In certain embodiments, compounds of Formula (XI) are stable in aqueoussolution for at least 1 month under mildly acidic conditions. In certainembodiments, compounds of Formula (XI) are stable for at least 2 weeksunder mildly acidic conditions. In certain embodiments, compound ofFormula (XI) are stable for at least 5 days under mildly acidicconditions. In certain embodiments, such acidic conditions are pH 2 to8.

Amino acids of Formula (XI) include amino acids having the structure ofFormula (XII):

wherein, B is optional, and when present is a linker selected from thegroup consisting of lower alkylene, substituted lower alkylene, loweralkenylene, substituted lower alkenylene, lower heteroalkylene,substituted lower heteroalkylene, —O—, —O-(alkylene or substitutedalkylene)-, —S—, —S-(alkylene or substituted alkylene)-, —S(O)_(k)—where k is 1, 2, or 3, —S(O)_(k)(alkylene or substituted alkylene)-,—C(O)—, —NS(O)₂—, —OS(O)₂—, —C(O)-(alkylene or substituted alkylene)-,—C(S)—, —C(S)-(alkylene or substituted alkylene)-, —N(R′)—,—NR′-(alkylene or substituted alkylene)-, —C(O)N(R′)—,—CON(R′)-(alkylene or substituted alkylene)-, —CSN(R′)—,—CSN(R′)-(alkylene or substituted alkylene)-, —N(R′)CO-(alkylene orsubstituted alkylene)-, —N(R′)C(O)O—, —S(O)_(k)N(R′)—, —N(R′)C(O)N(R′)—,—N(R′)C(S)N(R′)—, —N(R′)S(O)_(k)N(R′)—, —N(R′)—N═, —C(R′)═N—,—C(R′)═N—N(R′)—, —C(R′)═N—N═, —C(R′)₂—N═N—, and —C(R′)₂—N(R′)—N(R′)—,where each R′ is independently H, alkyl, or substituted alkyl; R is H,alkyl, substituted alkyl, cycloalkyl, or substituted cycloalkyl; R₁ isH, an amino protecting group, resin, amino acid, polypeptide, orpolynucleotide; and R₂ is OH, an ester protecting group, resin, aminoacid, polypeptide, or polynucleotide; R₅ is H, alkyl, substituted alkyl,alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, alkoxy,substituted alkoxy, alkylalkoxy, substituted alkylalkoxy, polyalkyleneoxide, substituted polyalkylene oxide, aryl, substituted aryl,heteroaryl, substituted heteroaryl, alkaryl, substituted alkaryl,aralkyl, substituted aralkyl, -(alkylene or substitutedalkylene)-ON(R″)₂, -(alkylene or substituted alkylene)-C(O)SR″,-(alkylene or substituted alkylene)-S—S-(aryl or substituted aryl),—C(O)R″, —C(O)₂R″, or —C(O)N(R″)₂, wherein each R″ is independentlyhydrogen, alkyl, substituted alkyl, alkenyl, substituted alkenyl,alkoxy, substituted alkoxy, aryl, substituted aryl, heteroaryl, alkaryl,substituted alkaryl, aralkyl, or substituted aralkyl; or R₅ is L-X,where X is a selected from the group consisting of a label; a dye; apolymer; a water-soluble polymer; a derivative of polyethylene glycol; aphotocrosslinker; a cytotoxic compound; a drug; an affinity label; aphotoaffinity label; a reactive compound; a resin; a second protein orpolypeptide or polypeptide analog; an antibody or antibody fragment; ametal chelator; a cofactor; a fatty acid; a carbohydrate; apolynucleotide; a DNA; a RNA; an antisense polynucleotide; a saccharide,a water-soluble dendrimer, a cyclodextrin, a biomaterial; ananoparticle; a spin label; a fluorophore, a metal-containing moiety; aradioactive moiety; a novel functional group; a group that covalently ornoncovalently interacts with other molecules; a photocaged moiety; aphotoisomerizable moiety; biotin; a biotin analogue; a moietyincorporating a heavy atom; a chemically cleavable group; aphotocleavable group; an elongated side chain; a carbon-linked sugar; aredox-active agent; an amino thioacid; a toxic moiety; an isotopicallylabeled moiety; a biophysical probe; a phosphorescent group; achemiluminescent group; an electron dense group; a magnetic group; anintercalating group; a chromophore; an energy transfer agent; abiologically active agent; a detectable label; and any combinationthereof; and L is optional, and when present is a linker selected fromthe group consisting of alkylene, substituted alkylene, alkenylene,substituted alkenylene, —O—, —O-(alkylene or substituted alkylene)-,—S—, —S-(alkylene or substituted alkylene)-, —S(O)_(k)— where k is 1, 2,or 3, —S(O)_(k)(alkylene or substituted alkylene)-, —C(O)—,—C(O)-(alkylene or substituted alkylene)-, —C(S)—, —C(S)-(alkylene orsubstituted alkylene)-, —N(R′)—, —NR′-(alkylene or substitutedalkylene)-, —C(O)N(R′)—, —CON(R′)-(alkylene or substituted alkylene)-,—CSN(R′)—, —CSN(R′)-(alkylene or substituted alkylene)-,—N(R′)CO-(alkylene or substituted alkylene)-, —N(R′)C(O)O—, -(alkyleneor substituted alkylene)-O—N═CR′—, -(alkylene or substitutedalkylene)-C(O)NR′-(alkylene or substituted alkylene)-, -(alkylene orsubstituted alkylene)-S(O)_(k)-(alkylene or substituted alkylene)-S—,-(alkylene or substituted alkylene)-S—S—, —S(O)_(k)N(R′)—,—N(R′)C(O)N(R′)—, —N(R′)C(S)N(R′)—, —N(R′)S(O)_(k)N(R′)—, —N(R′)—N═,—C(R′)═N—, —C(R′)═N—N(R′)—, —C(R′)═N—N═, —C(R′)₂—N═N—, and—C(R′)₂—N(R′)—N(R′)—, where each R′ is independently H, alkyl, orsubstituted alkyl. Such non-natural amino acids may be in the form of asalt, or may be incorporated into a non-natural amino acid polypeptide,polymer, polysaccharide, or a polynucleotide and optionally posttranslationally modified.

Such amino acids include amino acids having the structure of Formula(XIII):

wherein, R is H, alkyl, substituted alkyl, cycloalkyl, or substitutedcycloalkyl; R₁ is H, an amino protecting group, resin, amino acid,polypeptide, or polynucleotide; and R₂ is OH, an ester protecting group,resin, amino acid, polypeptide, or polynucleotide; R₅ is H, alkyl,substituted alkyl, alkenyl, substituted alkenyl, alkynyl, substitutedalkynyl, alkoxy, substituted alkoxy, alkylalkoxy, substitutedalkylalkoxy, polyalkylene oxide, substituted polyalkylene oxide, aryl,substituted aryl, heteroaryl, substituted heteroaryl, alkaryl,substituted alkaryl, aralkyl, substituted aralkyl, -(alkylene orsubstituted alkylene)-ON(R″)₂, -(alkylene or substitutedalkylene)-C(O)SR″, -(alkylene or substituted alkylene)-S—S-(aryl orsubstituted aryl), —C(O)R″, —C(O)₂R″, or —C(O)N(R″)₂, wherein each R″ isindependently hydrogen, alkyl, substituted alkyl, alkenyl, substitutedalkenyl, alkoxy, substituted alkoxy, aryl, substituted aryl, heteroaryl,alkaryl, substituted alkaryl, aralkyl, or substituted aralkyl; or R₅ isL-X, where X is a selected from the group consisting of a label; a dye;a polymer; a water-soluble polymer; a derivative of polyethylene glycol;a photocrosslinker; a cytotoxic compound; a drug; an affinity label; aphotoaffinity label; a reactive compound; a resin; a second protein orpolypeptide or polypeptide analog; an antibody or antibody fragment; ametal chelator; a cofactor; a fatty acid; a carbohydrate; apolynucleotide; a DNA; a RNA; an antisense polynucleotide; a saccharide,a water-soluble dendrimer, a cyclodextrin, a biomaterial; ananoparticle; a spin label; a fluorophore, a metal-containing moiety; aradioactive moiety; a novel functional group; a group that covalently ornoncovalently interacts with other molecules; a photocaged moiety; aphotoisomerizable moiety; biotin; a biotin analogue; a moietyincorporating a heavy atom; a chemically cleavable group; aphotocleavable group; an elongated side chain; a carbon-linked sugar; aredox-active agent; an amino thioacid; a toxic moiety; an isotopicallylabeled moiety; a biophysical probe; a phosphorescent group; achemiluminescent group; an electron dense group; a magnetic group; anintercalating group; a chromophore; an energy transfer agent; abiologically active agent; a detectable label; and any combinationthereof; and L is optional, and when present is a linker selected fromthe group consisting of alkylene, substituted alkylene, alkenylene,substituted alkenylene, —O—, —O-(alkylene or substituted alkylene)-,—S—, —S-(alkylene or substituted alkylene)-, —S(O)_(k)— where k is 1, 2,or 3, —S(O)_(k)(alkylene or substituted alkylene)-, —C(O)—,—C(O)-(alkylene or substituted alkylene)-, —C(S)—, —C(S)-(alkylene orsubstituted alkylene)-, —N(R′)—, —NR′-(alkylene or substitutedalkylene)-, —C(O)N(R′)—, —CON(R′)-(alkylene or substituted alkylene)-,—CSN(R′)—, —CSN(R′)-(alkylene or substituted alkylene)-,—N(R′)CO-(alkylene or substituted alkylene)-, —N(R′)C(O)O—, -(alkyleneor substituted alkylene)-O—N═CR′—, -(alkylene or substitutedalkylene)-C(O)NR′-(alkylene or substituted alkylene)-, -(alkylene orsubstituted alkylene)-S(O)_(k)-(alkylene or substituted alkylene)-S—,-(alkylene or substituted alkylene)-S—S—, —S(O)_(k)N(R′)—,—N(R′)C(O)N(R′)—, —N(R′)C(S)N(R′)—, —N(R′)S(O)_(k)N(R′)—, —N(R′)—N═,—C(R′)═N—, —C(R′)═N—N(R′)—, —C(R′)═N—N═, —C(R′)₂—N═N—, and—C(R′)₂—N(R′)—N(R′)—, where each R′ is independently H, alkyl, orsubstituted alkyl. Such non-natural amino acids may be in the form of asalt, or may be incorporated into a non-natural amino acid polypeptide,polymer, polysaccharide, or a polynucleotide and optionally posttranslationally modified.

Further non-limiting examples of such amino acids include amino acidshaving the following structures:

Such non-natural amino acids may be in the form of a salt, or may beincorporated into a non-natural amino acid polypeptide, polymer,polysaccharide, or a polynucleotide and optionally post translationallymodified.

In addition, such amino acids include amino acids having the structureof Formula (XIV):

wherein: A is optional, and when present is lower alkylene, substitutedlower alkylene, lower cycloalkylene, substituted lower cycloalkylene,lower alkenylene, substituted lower alkenylene, alkynylene, lowerheteroalkylene, substituted heteroalkylene, lower heterocycloalkylene,substituted lower heterocycloalkylene, arylene, substituted arylene,heteroarylene, substituted heteroarylene, alkarylene, substitutedalkarylene, aralkylene, or substituted aralkylene; B is optional, andwhen present is a linker selected from the group consisting of loweralkylene, substituted lower alkylene, lower alkenylene, substitutedlower alkenylene, lower heteroalkylene, substituted lowerheteroalkylene, —O—, —O-(alkylene or substituted alkylene)-, —S—,—S-(alkylene or substituted alkylene)-, —S(O)_(k)— where k is 1, 2, or3, —S(O)_(k)(alkylene or substituted alkylene)-, —C(O)—, —NS(O)₂—,—OS(O)₂—, —C(O)-(alkylene or substituted alkylene)-, —C(S)—,—C(S)-(alkylene or substituted alkylene)-, —N(R′)—, —NR′-(alkylene orsubstituted alkylene)-, —C(O)N(R′)—, —CON(R′)-(alkylene or substitutedalkylene)-, —CSN(R′)—, —CSN(R′)-(alkylene or substituted alkylene)-,—N(R′)CO-(alkylene or substituted alkylene)-, —N(R′)C(O)O—,—S(O)_(k)N(R′)—, —N(R′)C(O)N(R′)—, —N(R′)C(S)N(R′)—,—N(R′)S(O)_(k)N(R′)—, —N(R′)—N═, —C(R′)═N—, —C(R′)═N—N(R′)—,—C(R′)═N—N═, —C(R′)₂—N═N—, and —C(R′)₂—N(R′)—N(R′)—, where each R′ isindependently H, alkyl, or substituted alkyl; K is —NR₆R₇ or —N═CR₆R₇;R₁ is H, an amino protecting group, resin, amino acid, polypeptide, orpolynucleotide; and R₂ is OH, an ester protecting group, resin, aminoacid, polypeptide, or polynucleotide; each of R₃ and R₄ is independentlyH, halogen, lower alkyl, or substituted lower alkyl, or R₃ and R₄ or twoR₃ groups optionally form a cycloalkyl or a heterocycloalkyl; each of R₆and R₇ is independently selected from the group consisting of H, alkyl,substituted alkyl, alkenyl, substituted alkenyl, alkoxy, substitutedalkoxy, polyalkylene oxide, substituted polyalkylene oxide, aryl,substituted aryl, heteroaryl, substituted heteroaryl, alkaryl,substituted alkaryl, aralkyl, and substituted aralkyl, —C(O)R″,—C(O)₂R″, —C(O)N(R″)₂, wherein each R″ is independently hydrogen, alkyl,substituted alkyl, alkenyl, substituted alkenyl, alkoxy, substitutedalkoxy, aryl, substituted aryl, heteroaryl, alkaryl, substitutedalkaryl, aralkyl, or substituted aralkyl; or R₆ or R₇ is L-X, where X isa selected from the group consisting of a label; a dye; a polymer; awater-soluble polymer; a derivative of polyethylene glycol; aphotocrosslinker; a cytotoxic compound; a drug; an affinity label; aphotoaffinity label; a reactive compound; a resin; a second protein orpolypeptide or polypeptide analog; an antibody or antibody fragment; ametal chelator; a cofactor; a fatty acid; a carbohydrate; apolynucleotide; a DNA; a RNA; an antisense polynucleotide; a saccharide,a water-soluble dendrimer, a cyclodextrin, a biomaterial; ananoparticle; a spin label; a fluorophore, a metal-containing moiety; aradioactive moiety; a novel functional group; a group that covalently ornoncovalently interacts with other molecules; a photocaged moiety; aphotoisomerizable moiety; biotin; a biotin analogue; a moietyincorporating a heavy atom; a chemically cleavable group; aphotocleavable group; an elongated side chain; a carbon-linked sugar; aredox-active agent; an amino thioacid; a toxic moiety; an isotopicallylabeled moiety; a biophysical probe; a phosphorescent group; achemiluminescent group; an electron dense group; a magnetic group; anintercalating group; a chromophore; an energy transfer agent; abiologically active agent; a detectable label; and any combinationthereof; and L is optional, and when present is a linker selected fromthe group consisting of alkylene, substituted alkylene, alkenylene,substituted alkenylene, —O—, —O-(alkylene or substituted alkylene)-,—S—, —S-(alkylene or substituted alkylene)-, —S(O)_(k)— where k is 1, 2,or 3, —S(O)_(k)(alkylene or substituted alkylene)-, —C(O)—,—C(O)-(alkylene or substituted alkylene)-, —C(S)—, —C(S)-(alkylene orsubstituted alkylene)-, —N(R′)—, —NR′-(alkylene or substitutedalkylene)-, —C(O)N(R′)—, —CON(R′)-(alkylene or substituted alkylene)-,—CSN(R′)—, —CSN(R′)-(alkylene or substituted alkylene)-,—N(R′)CO-(alkylene or substituted alkylene)-, —N(R′)C(O)O—,—S(O)_(k)N(R′)—, —N(R′)C(O)N(R′)—, —N(R′)C(S)N(R′)—,—N(R′)S(O)_(k)N(R′)—, —N(R′)—N═, —C(R′)═N—, —C(R′)═N—N(R′)—,—C(R′)═N—N═, —C(R′)₂—N═N—, and —C(R′)₂—N(R′)—N(R′)—, where each R′ isindependently H, alkyl, or substituted alkyl. Such non-natural aminoacids may be in the form of a salt, or may be incorporated into anon-natural amino acid polypeptide, polymer, polysaccharide, or apolynucleotide and optionally post translationally modified.

Such amino acids further include amino acids having the structure ofFormula (XVI):

wherein: A is optional, and when present is lower alkylene, substitutedlower alkylene, lower cycloalkylene, substituted lower cycloalkylene,lower alkenylene, substituted lower alkenylene, alkynylene, lowerheteroalkylene, substituted heteroalkylene, lower heterocycloalkylene,substituted lower heterocycloalkylene, arylene, substituted arylene,heteroarylene, substituted heteroarylene, alkarylene, substitutedalkarylene, aralkylene, or substituted aralkylene; B is optional, andwhen present is a linker selected from the group consisting of loweralkylene, substituted lower alkylene, lower alkenylene, substitutedlower alkenylene, lower heteroalkylene, substituted lowerheteroalkylene, —O—, —O-(alkylene or substituted alkylene)-, —S—,—S-(alkylene or substituted alkylene)-, —S(O)_(k)— where k is 1, 2, or3, —S(O)_(k)(alkylene or substituted alkylene)-, —C(O)—, —NS(O)₂—,—OS(O)₂—, —C(O)-(alkylene or substituted alkylene)-, —C(S)—,—C(S)-(alkylene or substituted alkylene)-, —N(R′)—, —NR′-(alkylene orsubstituted alkylene)-, —C(O)N(R′)—, —CON(R′)-(alkylene or substitutedalkylene)-, —CSN(R′)—, —CSN(R′)-(alkylene or substituted alkylene)-,—N(R′)CO-(alkylene or substituted alkylene)-, —N(R′)C(O)O—,—S(O)_(k)N(R′)—, —N(R′)C(O)N(R′)—, —N(R′)C(S)N(R′)—,—N(R′)S(O)_(k)N(R′)—, —N(R′)—N═, —C(R′)═N—, —C(R′)═N—N(R′)—,—C(R′)═N—N═, —C(R′)₂—N═N—, and —C(R′)₂—N(R′)—N(R′)—, where each R′ isindependently H, alkyl, or substituted alkyl; R₁ is H, an aminoprotecting group, resin, amino acid, polypeptide, or polynucleotide; andR₂ is OH, an ester protecting group, resin, amino acid, polypeptide, orpolynucleotide; each of R₃ and R₄ is independently H, halogen, loweralkyl, or substituted lower alkyl, or R₃ and R₄ or two R₃ optionallyform a cycloalkyl or a heterocycloalkyl; each of R₆ and R₇ isindependently selected from the group consisting of H, alkyl,substituted alkyl, alkenyl, substituted alkenyl, alkoxy, substitutedalkoxy, polyalkylene oxide, substituted polyalkylene oxide, aryl,substituted aryl, heteroaryl, substituted heteroaryl, alkaryl,substituted alkaryl, aralkyl, and substituted aralkyl, —C(O)R″,—C(O)₂R″, —C(O)N(R″)₂, wherein each R″ is independently hydrogen, alkyl,substituted alkyl, alkenyl, substituted alkenyl, alkoxy, substitutedalkoxy, aryl, substituted aryl, heteroaryl, alkaryl, substitutedalkaryl, aralkyl, or substituted aralkyl; or R₆ or R₇ is L-X, where X isa selected from the group consisting of a label; a dye; a polymer; awater-soluble polymer; a derivative of polyethylene glycol; aphotocrosslinker; a cytotoxic compound; a drug; an affinity label; aphotoaffinity label; a reactive compound; a resin; a second protein orpolypeptide or polypeptide analog; an antibody or antibody fragment; ametal chelator; a cofactor; a fatty acid; a carbohydrate; apolynucleotide; a DNA; a RNA; an antisense polynucleotide; a saccharide,a water-soluble dendrimer, a cyclodextrin, a biomaterial; ananoparticle; a spin label; a fluorophore, a metal-containing moiety; aradioactive moiety; a novel functional group; a group that covalently ornoncovalently interacts with other molecules; a photocaged moiety; aphotoisomerizable moiety; biotin; a biotin analogue; a moietyincorporating a heavy atom; a chemically cleavable group; aphotocleavable group; an elongated side chain; a carbon-linked sugar; aredox-active agent; an amino thioacid; a toxic moiety; an isotopicallylabeled moiety; a biophysical probe; a phosphorescent group; achemiluminescent group; an electron dense group; a magnetic group; anintercalating group; a chromophore; an energy transfer agent; abiologically active agent; a detectable label; and any combinationthereof; and L is optional, and when present is a linker selected fromthe group consisting of alkylene, substituted alkylene, alkenylene,substituted alkenylene, —O—, —O-(alkylene or substituted alkylene)-,—S—, —S-(alkylene or substituted alkylene)-, —S(O)_(k)— where k is 1, 2,or 3, —S(O)_(k)(alkylene or substituted alkylene)-, —C(O)—,—C(O)-(alkylene or substituted alkylene)-, —C(S)—, —C(S)-(alkylene orsubstituted alkylene)-, —N(R′)—, —NR′-(alkylene or substitutedalkylene)-, —C(O)N(R′)—, —CON(R′)-(alkylene or substituted alkylene)-,—CSN(R′)—, —CSN(R′)-(alkylene or substituted alkylene)-,—N(R′)CO-(alkylene or substituted alkylene)-, —N(R′)C(O)O—,—S(O)_(k)N(R′)—, —N(R′)C(O)N(R′)—, —N(R′)C(S)N(R′)—,—N(R′)S(O)_(k)N(R′)—, —N(R′)—N═, —C(R′)═N—, —C(R′)═N—N(R′)—,—C(R′)═N—N═, —C(R′)₂—N═N—, and —C(R′)₂—N(R′)—N(R′)—, where each R′ isindependently H, alkyl, or substituted alkyl. Such non-natural aminoacids may be in the form of a salt, or may be incorporated into anon-natural amino acid polypeptide, polymer, polysaccharide, or apolynucleotide and optionally post translationally modified.

Further, such amino acids include amino acids having the structure ofFormula (XVII):

wherein: B is optional, and when present is a linker selected from thegroup consisting of lower alkylene, substituted lower alkylene, loweralkenylene, substituted lower alkenylene, lower heteroalkylene,substituted lower heteroalkylene, —O—, —O-(alkylene or substitutedalkylene)-, —S—, —S-(alkylene or substituted alkylene)-, —S(O)_(k)—where k is 1, 2, or 3, —S(O)_(k)(alkylene or substituted alkylene)-,—C(O)—, —NS(O)₂—, —OS(O)₂—, —C(O)-(alkylene or substituted alkylene)-,—C(S)—, —C(S)-(alkylene or substituted alkylene)-, —N(R′)—,—NR′-(alkylene or substituted alkylene)-, —C(O)N(R′)—,—CON(R′)-(alkylene or substituted alkylene)-, —CSN(R′)—,—CSN(R′)-(alkylene or substituted alkylene)-, —N(R′)CO-(alkylene orsubstituted alkylene)-, —N(R′)C(O)O—, —S(O)_(k)N(R′)—, —N(R′)C(O)N(R′)—,—N(R′)C(S)N(R′)—, —N(R′)S(O)_(k)N(R′)—, —N(R′)—N═, —C(R′)═N—,—C(R′)═N—N(R′)—, —C(R′)═N—N═, —C(R′)₂—N═N—, and —C(R′)₂—N(R′)—N(R′)—,where each R′ is independently H, alkyl, or substituted alkyl; R₁ is H,an amino protecting group, resin, amino acid, polypeptide, orpolynucleotide; and R₂ is OH, an ester protecting group, resin, aminoacid, polypeptide, or polynucleotide; each of R₆ and R₇ is independentlyselected from the group consisting of H, alkyl, substituted alkyl,alkenyl, substituted alkenyl, alkoxy, substituted alkoxy, polyalkyleneoxide, substituted polyalkylene oxide, aryl, substituted aryl,heteroaryl, substituted heteroaryl, alkaryl, substituted alkaryl,aralkyl, and substituted aralkyl, —C(O)R″, —C(O)₂R″, —C(O)N(R″)₂,wherein each R″ is independently hydrogen, alkyl, substituted alkyl,alkenyl, substituted alkenyl, alkoxy, substituted alkoxy, aryl,substituted aryl, heteroaryl, alkaryl, substituted alkaryl, aralkyl, orsubstituted aralkyl; or R₆ or R₇ is L-X, where X is a selected from thegroup consisting of a label; a dye; a polymer; a water-soluble polymer;a derivative of polyethylene glycol; a photocrosslinker; a cytotoxiccompound; a drug; an affinity label; a photoaffinity label; a reactivecompound; a resin; a second protein or polypeptide or polypeptideanalog; an antibody or antibody fragment; a metal chelator; a cofactor;a fatty acid; a carbohydrate; a polynucleotide; a DNA; a RNA; anantisense polynucleotide; a saccharide, a water-soluble dendrimer, acyclodextrin, a biomaterial; a nanoparticle; a spin label; afluorophore, a metal-containing moiety; a radioactive moiety; a novelfunctional group; a group that covalently or noncovalently interactswith other molecules; a photocaged moiety; a photoisomerizable moiety;biotin; a biotin analogue; a moiety incorporating a heavy atom; achemically cleavable group; a photocleavable group; an elongated sidechain; a carbon-linked sugar; a redox-active agent; an amino thioacid; atoxic moiety; an isotopically labeled moiety; a biophysical probe; aphosphorescent group; a chemiluminescent group; an electron dense group;a magnetic group; an intercalating group; a chromophore; an energytransfer agent; a biologically active agent; a detectable label; and anycombination thereof; and L is optional, and when present is a linkerselected from the group consisting of alkylene, substituted alkylene,alkenylene, substituted alkenylene, —O—, —O-(alkylene or substitutedalkylene)-, —S—, —S-(alkylene or substituted alkylene)-, —S(O)_(k)—where k is 1, 2, or 3, —S(O)_(k)(alkylene or substituted alkylene)-,—C(O)—, —C(O)-(alkylene or substituted alkylene)-, —C(S)—,—C(S)-(alkylene or substituted alkylene)-, —N(R′)—, —NR′-(alkylene orsubstituted alkylene)-, —C(O)N(R′)—, —CON(R′)-(alkylene or substitutedalkylene)-, —CSN(R′)—, —CSN(R′)-(alkylene or substituted alkylene)-,—N(R′)CO-(alkylene or substituted alkylene)-, —N(R′)C(O)O—,—S(O)_(k)N(R′)—, —N(R′)C(O)N(R′)—, —N(R′)C(S)N(R′)—,—N(R′)S(O)_(k)N(R′)—, —N(R′)—N═, —C(R′)═N—, —C(R′)═N—N(R′)—,—C(R′)═N—N═, —C(R′)₂—N═N—, and —C(R′)₂—N(R′)—N(R′)—, where each R′ isindependently H, alkyl, or substituted alkyl.

Non-limiting examples of such amino acids include amino acids having thefollowing structures:

Such non-natural amino acids may be in the form of a salt, or may beincorporated into a non-natural amino acid polypeptide, polymer,polysaccharide, or a polynucleotide and optionally post translationallymodified.

Additionally, such amino acids include amino acids having the structureof Formula (XVIII):

wherein: B is optional, and when present is a linker selected from thegroup consisting of lower alkylene, substituted lower alkylene, loweralkenylene, substituted lower alkenylene, lower heteroalkylene,substituted lower heteroalkylene, —O—, —O-(alkylene or substitutedalkylene)-, —S—, —S-(alkylene or substituted alkylene)-, —S(O)_(k)—where k is 1, 2, or 3, —S(O)_(k)(alkylene or substituted alkylene)-,—C(O)—, —NS(O)₂—, —OS(O)₂—, —C(O)-(alkylene or substituted alkylene)-,—C(S)—, —C(S)-(alkylene or substituted alkylene)-, —N(R′)—,—NR′-(alkylene or substituted alkylene)-, —C(O)N(R′)—,—CON(R′)-(alkylene or substituted alkylene)-, —CSN(R′)—,—CSN(R′)-(alkylene or substituted alkylene)-, —N(R′)CO-(alkylene orsubstituted alkylene)-, —N(R′)C(O)O—, —S(O)_(k)N(R′)—, —N(R′)C(O)N(R′)—,—N(R′)C(S)N(R′)—, —N(R′)S(O)_(k)N(R′)—, —N(R′)—N═, —C(R′)═N—,—C(R′)═N—N(R′)—, —C(R′)═N—N═, —C(R′)₂—N═N—, and —C(R′)₂—N(R′)—N(R′)—,where each R′ is independently H, alkyl, or substituted alkyl; R₁ is H,an amino protecting group, resin, amino acid, polypeptide, orpolynucleotide; and R₂ is OH, an ester protecting group, resin, aminoacid, polypeptide, or polynucleotide; each of R₆ and R₇ is independentlyselected from the group consisting of H, alkyl, substituted alkyl,alkenyl, substituted alkenyl, alkoxy, substituted alkoxy, polyalkyleneoxide, substituted polyalkylene oxide, aryl, substituted aryl,heteroaryl, substituted heteroaryl, alkaryl, substituted alkaryl,aralkyl, and substituted aralkyl, —C(O)R″, —C(O)₂R″, —C(O)N(R″)₂,wherein each R″ is independently hydrogen, alkyl, substituted alkyl,alkenyl, substituted alkenyl, alkoxy, substituted alkoxy, aryl,substituted aryl, heteroaryl, alkaryl, substituted alkaryl, aralkyl, orsubstituted aralkyl; or R₆ or R₇ is L-X, where X is a selected from thegroup consisting of a label; a dye; a polymer; a water-soluble polymer;a derivative of polyethylene glycol; a photocrosslinker; a cytotoxiccompound; a drug; an affinity label; a photoaffinity label; a reactivecompound; a resin; a second protein or polypeptide or polypeptideanalog; an antibody or antibody fragment; a metal chelator; a cofactor;a fatty acid; a carbohydrate; a polynucleotide; a DNA; a RNA; anantisense polynucleotide; a saccharide, a water-soluble dendrimer, acyclodextrin, a biomaterial; a nanoparticle; a spin label; afluorophore, a metal-containing moiety; a radioactive moiety; a novelfunctional group; a group that covalently or noncovalently interactswith other molecules; a photocaged moiety; a photoisomerizable moiety;biotin; a biotin analogue; a moiety incorporating a heavy atom; achemically cleavable group; a photocleavable group; an elongated sidechain; a carbon-linked sugar; a redox-active agent; an amino thioacid; atoxic moiety; an isotopically labeled moiety; a biophysical probe; aphosphorescent group; a chemiluminescent group; an electron dense group;a magnetic group; an intercalating group; a chromophore; an energytransfer agent; a biologically active agent; a detectable label; and anycombination thereof; and L is optional, and when present is a linkerselected from the group consisting of alkylene, substituted alkylene,alkenylene, substituted alkenylene, —O—, —O-(alkylene or substitutedalkylene)-, —S—, —S-(alkylene or substituted alkylene)-, —S(O)_(k)—where k is 1, 2, or 3, —S(O)_(k)(alkylene or substituted alkylene)-,—C(O)—, —C(O)-(alkylene or substituted alkylene)-, —C(S)—,—C(S)-(alkylene or substituted alkylene)-, —N(R′)—, —NR′-(alkylene orsubstituted alkylene)-, —C(O)N(R′)—, —CON(R′)-(alkylene or substitutedalkylene)-, —CSN(R′)—, —CSN(R′)-(alkylene or substituted alkylene)-,—N(R′)CO-(alkylene or substituted alkylene)-, —N(R′)C(O)O—,—S(O)_(k)N(R′)—, —N(R′)C(O)N(R′)—, —N(R′)C(S)N(R′)—,—N(R′)S(O)_(k)N(R′)—, —N(R′)—N═, —C(R′)═N—, —C(R′)═N—N(R′)—,—C(R′)═N—N═, —C(R′)₂—N═N—, and —C(R′)₂—N(R′)—N(R′)—, where each R′ isindependently H, alkyl, or substituted alkyl; and each R_(a) isindependently selected from the group consisting of H, halogen, alkyl,substituted alkyl, —N(R′)₂, —C(O)_(k)R′ where k is 1, 2, or 3,—C(O)N(R′)₂, —OR′, and —S(O)_(k)R′; where each R′ is independently H,alkyl, or substituted alkyl and n is 0 to 8. Such non-natural aminoacids may be in the form of a salt, or may be incorporated into anon-natural amino acid polypeptide, polymer, polysaccharide, or apolynucleotide and optionally post translationally modified.

Non-limiting examples of such amino acids include amino acids having thefollowing structures:

Such non-natural amino acids may be in the form of a salt, or may beincorporated into a non-natural amino acid polypeptide, polymer,polysaccharide, or a polynucleotide and optionally post translationallymodified.

In certain embodiments, the non-natural amino acid can be according toformula XIX:

or a salt thereof, wherein: D is —Ar—W₃— or —W₁—Y₁—C(O)—Y₂—W₂—; Ar is

each of W₁, W₂, and W₃ is independently a single bond or lower alkylene;each X₁ is independently —NH—, —O—, or —S—; each Y₁ is independently asingle bond, —NH—, or —O—; each Y₂ is independently a single bond, —NH—,—O—, or an N-linked or C-linked pyrrolidinylene; and one of Z₁, Z₂, andZ₃ is —N— and the others of Z₁, Z₂, and Z₃ are independently —CH—. Incertain embodiments, the non-natural amino acid is according to formulaXIXa:

where D is a defined in the context of formula XIX. In certainembodiments, the non-natural amino acid is according formula XIXb:

or a salt thereof, wherein W₄ is C₁-C₁₀ alkylene. In a furtherembodiment, W₄ is C₁-C₅ alkylene. In an embodiment, W₄ is C₁-C₃alkylene. In an embodiment, W₄ is C₁ alkylene. In particularembodiments, the non-natural amino acid is selected from the groupconsisting of:

or a salt thereof. Such non-natural amino acids may be in the form of asalt, or may be incorporated into a non-natural amino acid polypeptide,polymer, polysaccharide, or a polynucleotide and optionally posttranslationally modified.

Linkers and Payloads

In certain embodiments, the modified Fc protein comprises a non-naturalamino acid having a reactive group, as described above. One of skill inthe art can use the reactive group to link the modified Fc protein toany molecular entity capable of forming a covalent bond to thenon-natural amino acid, directly or indirectly via a linker.Accordingly, provided herein are conjugates of a modified Fc protein(also referred to as an Fc protein conjugate), as described herein,linked to one or more payload moieties via one or more non-natural aminoacids at site-specific positions. The payload can be linked directly orindirectly to the modified Fc protein, for instance, via a linker.

Any known methods for attaching therapeutic and/or diagnostic moleculesto an Fc domain or fragment can be used to attach a payload or conjugateto a modified Fc protein provided herein. For example, conventionalapproaches for chemical conjugation to the immunoglobulin Fc domaininclude random coupling to naturally occurring primary amines such aslysine and the amino-terminus or carboxylic acids such as glutamic acid,aspartic acid and the carboxy terminus. Alternatively, semi-selectivesite-specific coupling may be achieved through N-terminal conjugationunder appropriate conditions, or derivatized carbohydrates as found onFc proteins isolated from eukaryotic sources, or by partial reductionand coupling of native cysteine residues. (E.g., Kim et al., Apharmaceutical composition comprising an immunoglobulin Fc region as acarrier, WO 2005/047337). Additional information can be found in, forexample, U.S. Pat. No. 8,008,453 to Gegg et al., entitled “Modified Fcmolecules;” U.S. Pat. No. 7,887,809 to Garen et al., entitled“Neovascular-targeted immunoconjugates;” U.S. Pat. No. 8,124,094 to Kimet al., entitled “Immunoglobulin Fc fragment modified by non-peptidepolymer and pharmaceutical composition comprising the same;” Carter,2011, Experimental Cell Research, 317(9):1261-1269; Santi et al., 2012,PNAS 109(16): 6211-6216; and Reichert 2011, MAbs. 3(1): 76-99; each ofwhich is hereby incorporated by reference herein in its entirety.

Useful linkers include those described in the section above. In certainembodiments, the linker is any divalent or multivalent linker known tothose of skill in the art. Generally, the linker is capable of formingcovalent bonds to the functional moiety R and the alpha carbon of thenon-natural amino acid. Useful divalent linkers include a bond,alkylene, substituted alkylene, heteroalkylene, substitutedheteroalkylene, arylene, substituted arylene, heteroarlyene andsubstituted heteroarylene. In certain embodiments, the linker is C₁₋₁₀alkylene or C₁₋₁₀ heteroalkylene.

The molecular payload can be any molecular entity that one of skill inthe art might desire to conjugate to the Fc protein. In certainembodiments, the payload is a therapeutic moiety. In such embodiments,the Fc protein conjugate can be used to target the therapeutic moiety toits molecular target. In certain embodiments, the payload is a labelingmoiety. In such embodiments, the Fc protein conjugate can be used todetect binding of the Fc protein to its target. In certain embodiments,the payload is a cytotoxic moiety. In such embodiments, the conjugatecan be used target the cytotoxic moiety to a diseased cell, for example,a cancer cell, to initiate destruction or elimination of the cell.Conjugates comprising other molecular payloads apparent to those ofskill in the art are within the scope of the conjugates describedherein.

In certain embodiments, a conjugate can have a payload selected from thegroup consisting of a label, a dye, a polymer, a water-soluble polymer,polyethylene glycol, a derivative of polyethylene glycol, aphotocrosslinker, a cytotoxic compound, a radionuclide, a drug, anaffinity label, a photoaffinity label, a reactive compound, a resin, asecond protein or polypeptide or polypeptide analog, an antibody orantibody fragment, a metal chelator, a cofactor, a fatty acid, acarbohydrate, a polynucleotide, a DNA, a RNA, an antisensepolynucleotide, a peptide, a water-soluble dendrimer, a cyclodextrin, aninhibitory ribonucleic acid, a biomaterial, a nanoparticle, a spinlabel, a fluorophore, a metal-containing moiety, a radioactive moiety, anovel functional group, a group that covalently or noncovalentlyinteracts with other molecules, a photocaged moiety, a photoisomerizablemoiety, biotin, a derivative of biotin, a biotin analogue, a moietyincorporating a heavy atom, a chemically cleavable group, aphotocleavable group, an elongated side chain, a carbon-linked sugar, aredox-active agent, an amino thioacid, a toxic moiety, an isotopicallylabeled moiety, a biophysical probe, a phosphorescent group, achemiluminescent group, an electron dense group, a magnetic group, anintercalating group, a chromophore, an energy transfer agent, abiologically active agent, a detectable label, a small molecule, or anycombination thereof.

Useful drug payloads include any cytotoxic, cytostatic orimmunomodulatory drug. Useful classes of cytotoxic or immunomodulatoryagents include, for example, antitubulin agents, auristatins, DNA minorgroove binders, DNA replication inhibitors, alkylating agents (e.g.,platinum complexes such as cis-platin, mono(platinum), bis(platinum) andtri-nuclear platinum complexes and carboplatin), anthracyclines,antibiotics, antifolates, antimetabolites, calmodulin inhibitors,chemotherapy sensitizers, duocarmycins, etoposides, fluorinatedpyrimidines, ionophores, lexitropsins, maytansinoids, nitrosoureas,platinols, pore-forming compounds, purine antimetabolites, puromycins,radiation sensitizers, rapamycins, steroids, taxanes, topoisomeraseinhibitors, vinca alkaloids, or the like.

Individual cytotoxic or immunomodulatory agents include, for example, anandrogen, anthramycin (AMC), asparaginase, 5-azacytidine, azathioprine,bleomycin, busulfan, buthionine sulfoximine, calicheamicin,calicheamicin derivatives, camptothecin, carboplatin, carmustine (BSNU),CC-1065, chlorambucil, cisplatin, colchicine, cyclophosphamide,cytarabine, cytidine arabinoside, cytochalasin B, dacarbazine,dactinomycin (formerly actinomycin), daunorubicin, decarbazine, DM1,DM4, docetaxel, doxorubicin, etoposide, an estrogen,5-fluordeoxyuridine, 5-fluorouracil, gemcitabine, gramicidin D,hydroxyurea, idarubicin, ifosfamide, irinotecan, lomustine (CCNU),maytansine, mechlorethamine, melphalan, 6-mercaptopurine, methotrexate,mithramycin, mitomycin C, mitoxantrone, nitroimidazole, paclitaxel,palytoxin, plicamycin, procarbizine, rhizoxin, streptozotocin,tenoposide, 6-thioguanine, thioTEPA, topotecan, vinblastine,vincristine, vinorelbine, VP-16 and VM-26.

In some embodiments, suitable cytotoxic agents include, for example, DNAminor groove binders (e.g., enediynes and lexitropsins, a CBI compound;see also U.S. Pat. No. 6,130,237), duocarmycins, taxanes (e.g.,paclitaxel and docetaxel), puromycins, vinca alkaloids, CC-1065, SN-38,topotecan, morpholino-doxorubicin, rhizoxin,cyanomorpholino-doxorubicin, echinomycin, combretastatin, netropsin,epothilone A and B, estramustine, cryptophycins, cemadotin,maytansinoids, discodermolide, eleutherobin, and mitoxantrone.

In some embodiments, the payload is one or more therapeutic peptides.Any peptide that exhibits a therapeutic effect can be included as apayload to a modified Fc protein. The therapeutic peptides can be of anylength; for example, a therapeutic peptide can have two or more aminoacids, three or more amino acids, four or more amino acids, five or moreamino acids, six or more amino acids, seven or more amino acids, eightor more amino acids, nine or more amino acids, 10 or more amino acids,11 or more amino acids, 12 or more amino acids, 13 or more amino acids,14 or more amino acids, 15 or more amino acids, 16 or more amino acids,17 or more amino acids, 18 or more amino acids, 19 or more amino acids,20 or more amino acids, 22 or more amino acids, 24 or more amino acids,26 or more amino acids, 28 or more amino acids, 30 or more amino acids,32 or more amino acids, 34 or more amino acids, 36 or more amino acids,38 or more amino acids, 40 or more amino acids, 42 or more amino acids,44 or more amino acids, 46 or more amino acids, 48 or more amino acids,50 or more amino acids, 55 or more amino acids, 60 or more amino acids,65 or more amino acids, 70 or more amino acids, 75 or more amino acids,80 or more amino acids, 85 or more amino acids, 90 or more amino acids,95 or more amino acids, 100 or more amino acids, 110 or more aminoacids, 120 or more amino acids, 130 or more amino acids, 140 or moreamino acids, 150 or more amino acids, 160 or more amino acids, 170 ormore amino acids, 180 or more amino acids, 190 or more amino acids, 140or more amino acids, 150 or more amino acids, 160 or more amino acids,170 or more amino acids, 180 or more amino acids, 190 or more aminoacids, 200 or more amino acids, 220 or more amino acids, 250 or moreamino acids, 275 or more amino acids, 300 or more amino acids, 350 ormore amino acids, 400 or more amino acids, 450 or more amino acids, or500 or more amino acids.

In some embodiments, the therapeutic peptide is a fragment of a knownprotein. For example, the therapeutic peptide can have two or more aminoacids, three or more amino acids, four or more amino acids, five or moreamino acids, six or more amino acids, seven or more amino acids, eightor more amino acids, nine or more amino acids, 10 or more amino acids,11 or more amino acids, 12 or more amino acids, 13 or more amino acids,14 or more amino acids, 15 or more amino acids, 16 or more amino acids,17 or more amino acids, 18 or more amino acids, 19 or more amino acids,20 or more amino acids, 22 or more amino acids, 24 or more amino acids,26 or more amino acids, 28 or more amino acids, 30 or more amino acids,32 or more amino acids, 34 or more amino acids, 36 or more amino acids,38 or more amino acids, 40 or more amino acids, 42 or more amino acids,44 or more amino acids, 46 or more amino acids, 48 or more amino acids,50 or more amino acids, 55 or more amino acids, 60 or more amino acids,65 or more amino acids, 70 or more amino acids, 75 or more amino acids,80 or more amino acids, 85 or more amino acids, 90 or more amino acids,95 or more amino acids, 100 or more amino acids, 110 or more aminoacids, 120 or more amino acids, 130 or more amino acids, 140 or moreamino acids, 150 or more amino acids, 160 or more amino acids, 170 ormore amino acids, 180 or more amino acids, 190 or more amino acids, 140or more amino acids, 150 or more amino acids, 160 or more amino acids,170 or more amino acids, 180 or more amino acids, 190 or more aminoacids, 200 or more amino acids, 220 or more amino acids, 250 or moreamino acids, 275 or more amino acids, 300 or more amino acids, 350 ormore amino acids, 400 or more amino acids, 450 or more amino acids, or500 or more amino acids from a contiguous fragment of a known protein.

In some embodiments, the sequence of a therapeutic peptide is based onand shared sequence identity with or similarity to a known protein. Insome embodiments, the therapeutic peptide has at least about 50%, 51%,52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60% or greater sequence identitywith the amino acid sequence of a contiguous fragment of a knownprotein.

In some embodiments, the therapeutic peptide has at least about 61%,62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70% or greater sequence identitywith the amino acid sequence of a contiguous fragment of a knownprotein.

In some embodiments, the therapeutic peptide has at least about 71%,72%, 73%, 74%, 75%, 76%, 77%, 78%, 79% or 80% or greater sequenceidentity with the amino acid sequence of a contiguous fragment of aknown protein.

In some embodiments, the therapeutic peptide has at least about 81%,82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, or 90% or greater sequenceidentity with the amino acid sequence of a contiguous fragment of aknown protein.

In some embodiments, the therapeutic peptide has at least about t 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or greater sequence identitywith the amino acid sequence of a contiguous fragment of a knownprotein.

In some embodiments, the therapeutic peptide can include any non-naturalor modified amino acid residues known in the art. In advantageousembodiments, the non-natural or modified amino acid facilitates linkageto the modified Fc protein directly via a covalent bond or indirectlyvia a linker.

Exemplary non-natural amino acids and amino acid analogs that can beused in the therapeutic peptides include, but are not limited to,2-aminobutyric acid, 2-aminoisobutyric acid, 3-(1-naphthyl)alanine,3-(2-naphthyl)alanine, 3-methylhistidine, 3-pyridylalanine,4-chlorophenylalanine, 4-fluorophenylalanine, 4-hydroxyproline,5-hydroxylysine, alloisoleucine, citrulline, dehydroalanine,homoarginine, homocysteine, homoserine, hydroxyproline, N-acetylserine,N-formylmethionine, N-methylglycine, N-methylisoleucine, norleucine,N-alpha.-methylarginine, O-phosphoserine, ornithine, phenylglycine,pipecolinic acid, piperazic acid, pyroglutamine, sarcosine, valanine,β-alanine, and β-cyclohexylalanine. Further useful amino acids includethose described herein.

In certain embodiments, therapeutic peptides can be a cytokine, a growthfactor, a factor for regulating replication, transcription, ortranslation, or a homolog or analog thereof. In particular embodiments,exemplary therapeutic peptides include but are not limited toadrenomedullin (AM), argatroban, angiopoietin (Ang), autocrine motilityfactor, bone morphogenetic proteins (BMPs), brain-derived neurotrophicfactor (BDNF), ciliary neurotrophic factor (CNTF), elafin, epidermalgrowth factor (EGF), erythropoietin (EPO), exendin-3, exendin-4,fibroblast growth factor (FGF), Glial cell line-derived neurotrophicfactor (GDNF), gonadotropin, granulocyte colony-stimulating factor(G-CSF), granulocyte macrophage colony-stimulating factor (GM-CSF),growth differentiation factor-9 (GDF9), hepatocyte growth factor (HGF),hepatoma-derived growth factor (HDGF), insulin-like growth factor (IGF),interferon (IFN), migration-stimulating factor, myostatin (GDF-8), nervegrowth factor (NGF) and other neurotrophins, platelet-derived growthfactor (PDGF), prolactin, thrombopoietin (TPO), somatotropin,transforming growth factor alpha (TGF-α), transforming growth factorbeta (TGF-β), tumor necrosis factor-alpha (TNF-α), vascular endothelialgrowth factor (VEGF), a wnt Signaling Pathway regulator of homologthereof, placental growth factor (P1GF), fetal Bovine Somatotrophin(FBS), X-Linked inhibitor of apoptosis protein (XIAP), or an interleukinsuch as IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10,viral IL-10, IL-11, IL-12, IL-13, IL-14, IL-15, IL-16, IL-17, and IL-18,

Additional exemplary therapeutic peptides and methods for making thesame are described in, for example, United States Patent Publication No.2007/0003518 by Atkinson et al.; United States Patent Publication No.2010/0285003 by Eggink et al.; United States Patent Publication No.2004/0067888 by Tovey et al.; United States Patent Publication No.20050267028 by M. Virji; and United States Patent Publication No.2011/0166063 by Bossard et al.; each of which is hereby incorporated byreference herein in its entirety.

Conjugates of therapeutic peptides and modified Fc proteins can beprepared using any method known in the art and described herein. Forexample, in some embodiments, a therapeutic peptide can be attached toany non-natural amino acid of an Fc protein as described herein. In someembodiments, a therapeutic peptide containing a non-natural amino acidcan be linked to a modified Fc protein at a non-natural amino acid ofthe Fc protein as described herein. Further, in some embodiments,modified Fc proteins can be coupled to any other reactive group, forinstance, an amino, carboxy or thiol group, of a therapeutic peptidestandard coupling reagents or linkers.

In some embodiments, a targeting moiety is included in an Fc proteinconjugate to guide the therapeutic or diagnostic payload to a desiredlocation within a subject. The term “targeting moiety” is used herein torefer to a molecular structure that helps the conjugates of theinvention to localize to a targeting area, e.g., help enter a cell, orbind a receptor. Preferably, the targeting moiety comprises a vitamin,antibody, antigen, receptor, DNA, RNA, sialyl Lewis X antigen,hyaluronic acid, sugars, cell specific lectins, steroid or steroidderivative, RGD peptide, ligand for a cell surface receptor, serumcomponent, or combinatorial molecule directed against various intra- orextracellular receptors. The targeting moiety can also comprise a lipidor a phospholipid. Exemplary phospholipids include, without limitation,phosphatidylcholines, phospatidylserine, phospatidylinositol,phospatidylglycerol, and phospatidylethanolamine. These lipids may be inthe form of micelles or liposomes and the like. The targeting moiety mayfurther comprise a detectable label or alternately a detectable labelmay serve as a targeting moiety. When the conjugate has a targetinggroup comprising a detectable label, the amount and/ordistribution/location of the polymer and/or the moiety (e.g., activeagent) to which the polymer is coupled can be determined by using asuitable detector. Such labels include, without limitation, fluorescers,chemiluminescers, moieties used in enzyme labeling, colorimetric (e.g.,dyes), metal ions, radioactive moieties, gold particles, quantum dots,and the like.

In some embodiments, the payload is an anti-tubulin agent. Examples ofanti-tubulin agents include, but are not limited to, taxanes (e.g.,Taxol® (paclitaxel), Taxotere® (docetaxel)), T67 (Tularik) and vincaalkyloids (e.g., vincristine, vinblastine, vindesine, and vinorelbine).Other antitubulin agents include, for example, baccatin derivatives,taxane analogs, epothilones (e.g., epothilone A and B), nocodazole,colchicine and colcimid, estramustine, cryptophycins, cemadotin,maytansinoids, combretastatins, discodermolide, and eleutherobin.

In certain embodiments, the cytotoxic agent is a maytansinoid, anothergroup of anti-tubulin agents. For example, in specific embodiments, themaytansinoid can be maytansine or DM-1 (ImmunoGen, Inc.; see also Chariet al., 1992, Cancer Res. 52:127-131).

In some embodiments, the payload is an auristatin, such as auristatin Eor a derivative thereof. For example, the auristatin E derivative can bean ester formed between auristatin E and a keto acid. For example,auristatin E can be reacted with paraacetyl benzoic acid orbenzoylvaleric acid to produce AEB and AEVB, respectively. Other typicalauristatin derivatives include AFP, MMAF, and MMAE. The synthesis andstructure of auristatin derivatives are described in U.S. PatentApplication Publication Nos. 2003-0083263, 2005-0238649 and2005-0009751; International Patent Publication No. WO 04/010957,International Patent Publication No. WO 02/088172, and U.S. Pat. Nos.6,323,315; 6,239,104; 6,034,065; 5,780,588; 5,665,860; 5,663,149;5,635,483; 5,599,902; 5,554,725; 5,530,097; 5,521,284; 5,504,191;5,410,024; 5,138,036; 5,076,973; 4,986,988; 4,978,744; 4,879,278;4,816,444; and 4,486,414.

In some embodiments, the payload comprises a radioisotope. In someembodiments, the payload does not comprise a radioisotope. In someembodiments, the payload is radioactive. In some embodiments, thepayload is not radioactive. In some embodiments, the payload comprises alabel or tag, created based on fluorescence (e.g., green or redfluorescent protein), resonance, luminescence (e.g., bioluminescence,chemiluminescence, electrochemiluminescence, crystalloluminescence,produced during crystallization, electroluminescence,cathodoluminescence, mechanoluminescence, triboluminescence,fractoluminescence, piezoluminescence, photoluminescence, fluorescence,phosphorescence, radioluminescence, a result of bombardment by ionizingradiation, sonoluminescence, thermoluminescence), chelation, metal(e.g., gold particles), quantum dots, moieties used in enzyme labeling,colorimetric (e.g., dyes), or any specific molecular bindinginteractions.

In some embodiments, when used for diagnostic or labeling purposes, thepayload can be a non-biological moiety. In some embodiments, the payloadcan be a non-biological moiety attached to a biological moiety such as apeptide, a protein, a nucleic acid or a hybrid thereof.

In some embodiments, the payload is an antimetabolite. Theantimetabolite can be, for example, a purine antagonist (e.g.,azothioprine or mycophenolate mofetil), a dihydrofolate reductaseinhibitor (e.g., methotrexate), acyclovir, ganciclovir, zidovudine,vidarabine, ribavarin, azidothymidine, cytidine arabinoside, amantadine,dideoxyuridine, iododeoxyuridine, poscarnet, or trifluridine.

In other embodiments, the payload is tacrolimus, cyclosporine, FU506 orrapamycin. In further embodiments, the payload is a drug such asaldesleukin, alemtuzumab, alitretinoin, allopurinol, altretamine,amifostine, anastrozole, arsenic trioxide, bexarotene, bexarotene,calusterone, capecitabine, celecoxib, cladribine, Darbepoetin alfa,Denileukin diftitox, dexrazoxane, dromostanolone propionate, epirubicin,Epoetin alfa, estramustine, exemestane, Filgrastim, floxuridine,fludarabine, fulvestrant, gemcitabine, gemtuzumab ozogamicin(MYLOTARG®), goserelin, idarubicin, ifosfamide, imatinib mesylate,Interferon alfa-2a, irinotecan, letrozole, leucovorin, levamisole,meclorethamine or nitrogen mustard, megestrol, mesna, methotrexate,methoxsalen, mitomycin C, mitotane, nandrolone phenpropionate,oprelvekin, oxaliplatin, pamidronate, pegademase, pegaspargase,pegfilgrastim, pentostatin, pipobroman, plicamycin, porfimer sodium,procarbazine, quinacrine, rasburicase, Rituximab, Sargramostim,streptozocin, tamoxifen, temozolomide, teniposide, testolactone,thioguanine, toremifene, Tositumomab, Trastuzumab (HERCEPTIN®),tretinoin, uracil mustard, valrubicin, vinblastine, vincristine,vinorelbine or zoledronate.

In some embodiments, the payload is an immunomodulatory agent. Theimmunomodulatory agent can be, for example, ganciclovir, etanercept,tacrolimus, cyclosporine, rapamycin, cyclophosphamide, azathioprine,mycophenolate mofetil or methotrexate. Alternatively, theimmunomodulatory agent can be, for example, a glucocorticoid (e.g.,cortisol or aldosterone) or a glucocorticoid analogue (e.g., prednisoneor dexamethasone).

In some embodiments, the immunomodulatory agent is an anti-inflammatoryagent, such as arylcarboxylic derivatives, pyrazole-containingderivatives, oxicam derivatives and nicotinic acid derivatives. Classesof anti-inflammatory agents include, for example, cyclooxygenaseinhibitors, 5-lipoxygenase inhibitors, and leukotriene receptorantagonists.

Suitable cyclooxygenase inhibitors include meclofenamic acid, mefenamicacid, carprofen, diclofenac, diflunisal, fenbufen, fenoprofen,indomethacin, ketoprofen, nabumetone, sulindac, tenoxicam and tolmetin.

Suitable lipoxygenase inhibitors include redox inhibitors (e.g.,catechol butane derivatives, nordihydroguaiaretic acid (NDGA),masoprocol, phenidone, lanopalen, indazolinones, naphazatrom,benzofuranol, alkylhydroxylamine), and non-redox inhibitors (e.g.,hydroxythiazoles, methoxyalkylthiazoles, benzopyrans and derivativesthereof, methoxytetrahydropyran, boswellic acids and acetylatedderivatives of boswellic acids, and quinolinemethoxyphenylacetic acidssubstituted with cycloalkyl radicals), and precursors of redoxinhibitors.

Other suitable lipoxygenase inhibitors include antioxidants (e.g.,phenols, propyl gallate, flavonoids and/or naturally occurringsubstrates containing flavonoids, hydroxylated derivatives of theflavones, flavonol, dihydroquercetin, luteolin, galangin, orobol,derivatives of chalcone, 4,2′,4′-trihydroxychalcone, ortho-aminophenols,N-hydroxyureas, benzofuranols, ebselen and species that increase theactivity of the reducing selenoenzymes), iron chelating agents (e.g.,hydroxamic acids and derivatives thereof, N-hydroxyureas,2-benzyl-1-naphthol, catechols, hydroxylamines, camosol trolox C,catechol, naphthol, sulfasalazine, zyleuton, 5-hydroxyanthranilic acidand 4-(omega-arylalkyl)phenylalkanoic acids), imidazole-containingcompounds (e.g., ketoconazole and itraconazole), phenothiazines, andbenzopyran derivatives.

Yet other suitable lipoxygenase inhibitors include inhibitors ofeicosanoids (e.g., octadecatetraenoic, eicosatetraenoic,docosapentaenoic, eicosahexaenoic and docosahexaenoic acids and estersthereof, PGE1 (prostaglandin E1), PGA2 (prostaglandin A2), viprostol,15-monohydroxyeicosatetraenoic, 15-monohydroxy-eicosatrienoic and15-monohydroxyeicosapentaenoic acids, and leukotrienes B5, C5 and D5),compounds interfering with calcium flows, phenothiazines,diphenylbutylamines, verapamil, fuscoside, curcumin, chlorogenic acid,caffeic acid, 5,8,11,14-eicosatetrayenoic acid (ETYA),hydroxyphenylretinamide, Ionapalen, esculin, diethylcarbamazine,phenantroline, baicalein, proxicromil, thioethers, diallyl sulfide anddi-(1-propenyl)sulfide.

Leukotriene receptor antagonists include calcitriol, ontazolast, BayerBay-x-1005, Ciba-Geigy CGS-25019C, ebselen, Leo Denmark ETH-615, LillyLY-293111, Ono ONO-4057, Terumo TMK-688, Boehringer Ingleheim BI-RM-270,Lilly LY 213024, Lilly LY 264086, Lilly LY 292728, Ono ONO LB457, Pfizer105696, Perdue Frederick PF 10042, Rhone-Poulenc Rorer RP 66153,SmithKline Beecham SB-201146, SmithKline Beecham SB-201993, SmithKlineBeecham SB-209247, Searle SC-53228, Sumitamo SM 15178, American HomeProducts WAY 121006, Bayer Bay-o-8276, Warner-Lambert CI-987,Warner-Lambert CI-987BPC-15LY 223982, Lilly LY 233569, Lilly LY-255283,MacroNex MNX-160, Merck and Co. MK-591, Merck and Co. MK-886, OnoONO-LB-448, Purdue Frederick PF-5901, Rhone-Poulenc Rorer RG14893,Rhone-Poulenc Rorer RP 66364, Rhone-Poulenc Rorer RP 69698, ShionoogiS-2474, Searle SC-41930, Searle SC-50505, Searle SC-51146, SearleSC-52798, SmithKline Beecham SK&F-104493, Leo Denmark SR-2566, TanabeT-757 and Teijin TEI-1338.

Other useful drug payloads include chemical compounds useful in thetreatment of cancer. Examples of chemotherapeutic agents includeErlotinib (TARCEVA®, Genentech/OSI Pharm.), Bortezomib (VELCADE®,Millennium Pharm.), Fulvestrant (FASLODEX®, AstraZeneca), Sutent(SU11248, Pfizer), Letrozole (FEMARA®, Novartis), Imatinib mesylate(GLEEVEC®, Novartis), PTK787/ZK 222584 (Novartis), Oxaliplatin(Eloxatin®, Sanofi), 5-FU (5-fluorouracil), Leucovorin, Rapamycin(Sirolimus, RAPAMUNE®, Wyeth), Lapatinib (TYKERB®, GSK572016, GlaxoSmith Kline), Lonafamib (SCH 66336), Sorafenib (BAY43-9006, Bayer Labs),and Gefitinib (IRESSA®, AstraZeneca), AG1478, AG1571 (SU 5271; Sugen),alkylating agents such as thiotepa and CYTOXAN® cyclosphosphamide; alkylsulfonates such as busulfan, improsulfan and piposulfan; aziridines suchas benzodopa, carboquone, meturedopa, and uredopa; ethylenimines andmethylamelamines including altretamine, triethylenemelamine,triethylenephosphoramide, triethylenethiophosphoramide andtrimethylomelamine; acetogenins (especially bullatacin andbullatacinone); a camptothecin (including the synthetic analogtopotecan); bryostatin; callystatin; CC-1065 (including its adozelesin,carzelesin and bizelesin synthetic analogs); cryptophycins (particularlycryptophycin 1 and cryptophycin 8); dolastatin; duocarmycin (includingthe synthetic analogs, KW-2189 and CB1-TM1); eleutherobin;pancratistatin; a sarcodictyin; spongistatin; nitrogen mustards such aschlorambucil, chlomaphazine, chlorophosphamide, estramustine,ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride,melphalan, novembichin, phenesterine, prednimustine, trofosfamide,uracil mustard; nitrosureas such as carmustine, chlorozotocin,fotemustine, lomustine, nimustine, and ranimnustine; antibiotics such asthe enediyne antibiotics (e.g., calicheamicin, especially calicheamicingammall and calicheamicin omegall (Angew Chem. Intl. Ed. Engl. (1994)33:183-186); dynemicin, including dynemicin A; bisphosphonates, such asclodronate; an esperamicin; as well as neocarzinostatin chromophore andrelated chromoprotein enediyne antibiotic chromophores), aclacinomysins,actinomycin, authramycin, azaserine, bleomycins, cactinomycin,carabicin, caminomycin, carzinophilin, chromomycinis, dactinomycin,daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine, ADRIAMYCIN®(doxorubicin), morpholino-doxorubicin, cyanomorpholino-doxorubicin,2-pyrrolino-doxorubicin and deoxydoxorubicin), epirubicin, esorubicin,idarubicin, marcellomycin, mitomycins such as mitomycin C, mycophenolicacid, nogalamycin, olivomycins, peplomycin, porfiromycin, puromycin,quelamycin, rodorubicin, streptonigrin, streptozocin, tubercidin,ubenimex, zinostatin, zorubicin; anti-metabolites such as methotrexateand 5-fluorouracil (5-FU); folic acid analogs such as denopterin,methotrexate, pteropterin, trimetrexate; purine analogs such asfludarabine, 6-mercaptopurine, thiamniprine, thioguanine; pyrimidineanalogs such as ancitabine, azacitidine, 6-azauridine, carmofur,cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine;androgens such as calusterone, dromostanolone propionate, epitiostanol,mepitiostane, testolactone; anti-adrenals such as aminoglutethimide,mitotane, trilostane; folic acid replenisher such as frolinic acid;aceglatone; aldophosphamide glycoside; aminolevulinic acid; eniluracil;amsacrine; bestrabucil; bisantrene; edatraxate; defofamine; demecolcine;diaziquone; elformithine; elliptinium acetate; an epothilone; etoglucid;gallium nitrate; hydroxyurea; lentinan; lonidainine; maytansinoids suchas maytansine and ansamitocins; mitoguazone; mitoxantrone; mopidanmol;nitraerine; pentostatin; phenamet; pirarubicin; losoxantrone;podophyllinic acid; 2-ethylhydrazide; procarbazine; PSK® polysaccharidecomplex (JHS Natural Products, Eugene, Oreg.); razoxane; rhizoxin;sizofuran; spirogermanium; tenuazonic acid; triaziquone;2,2′,2″-trichlorotriethylamine; trichothecenes (especially T-2 toxin,verracurin A, roridin A and anguidine); urethan; vindesine; dacarbazine;mannomustine; mitobronitol; mitolactol; pipobroman; gacytosine;arabinoside (“Ara-C”); cyclophosphamide; thiotepa; taxoids, e.g., TAXOL®(paclitaxel; Bristol-Myers Squibb Oncology, Princeton, N.J.), ABRAXANE®(Cremophor-free), albumin-engineered nanoparticle formulations ofpaclitaxel (American Pharmaceutical Partners, Schaumberg, Ill.), andTAXOTERE® (doxetaxel; Rhone-Poulenc Rorer, Antony, France);chloranmbucil; GEMZAR® (gemcitabine); 6-thioguanine; mercaptopurine;methotrexate; platinum analogs such as cisplatin and carboplatin;vinblastine; etoposide (VP-16); ifosfamide; mitoxantrone; vincristine;NAVELBINE® (vinorelbine); novantrone; teniposide; edatrexate;daunomycin; aminopterin; capecitabine (XELODA®); ibandronate; CPT-11;topoisomerase inhibitor RFS 2000; difluoromethylornithine (DMFO);retinoids such as retinoic acid; and pharmaceutically acceptable salts,acids and derivatives of any of the above.

Other useful payloads include: (i) anti-hormonal agents that act toregulate or inhibit hormone action on tumors such as anti-estrogens andselective estrogen receptor modulators (SERMs), including, for example,tamoxifen (including NOLVADEX®; tamoxifen citrate), raloxifene,droloxifene, 4-hydroxytamoxifen, trioxifene, keoxifene, LY117018,onapristone, and FARESTON® (toremifine citrate); (ii) aromataseinhibitors that inhibit the enzyme aromatase, which regulates estrogenproduction in the adrenal glands, such as, for example, 4(5)-imidazoles,aminoglutethimide, MEGASE® (megestrol acetate), AROMASIN® (exemestane;Pfizer), formestanie, fadrozole, RIVISOR® (vorozole), FEMARA®(letrozole; Novartis), and ARIMIDEX® (anastrozole; AstraZeneca); (iii)anti-androgens such as flutamide, nilutamide, bicalutamide, leuprolide,and goserelin; as well as troxacitabine (a 1,3-dioxolane nucleosidecytosine analog); (iv) protein kinase inhibitors; (v) lipid kinaseinhibitors; (vi) antisense oligonucleotides, particularly those whichinhibit expression of genes in signaling pathways implicated in aberrantcell proliferation, such as, for example, PKC-alpha, Ralf and H-Ras;(vii) ribozymes such as VEGF expression inhibitors (e.g., ANGIOZYME®)and HER2 expression inhibitors; (viii) vaccines such as gene therapyvaccines, for example, ALLOVECTIN®, LEUVECTIN®, and VAXID®; PROLEUKIN®rIL-2; a topoisomerase 1 inhibitor such as LURTOTECAN®; ABARELIX® rmRH;(ix) anti-angiogenic agents such as bevacizumab (AVASTIN®, Genentech);and (x) pharmaceutically acceptable salts, acids and derivatives of anyof the above. Other anti-angiogenic agents include MMP-2(matrix-metalloproteinase 2) inhibitors, MMP-9 (matrix-metalloproteinase9) inhibitors, COX-II (cyclooxygenase II) inhibitors, and VEGF receptortyrosine kinase inhibitors. Examples of such useful matrixmetalloproteinase inhibitors that can be used in combination with thepresent compounds/compositions are described in WO 96/33172, WO96/27583, EP 818442, EP 1004578, WO 98/07697, WO 98/03516, WO 98/34918,WO 98/34915, WO 98/33768, WO 98/30566, EP 606,046, EP 931,788, WO90/05719, WO 99/52910, WO 99/52889, WO 99/29667, WO 99/07675, EP 945864,U.S. Pat. No. 5,863,949, U.S. Pat. No. 5,861,510, and EP 780,386, all ofwhich are incorporated herein in their entireties by reference. Examplesof VEGF receptor tyrosine kinase inhibitors include4-(4-bromo-2-fluoroanilino)-6-methoxy-7-(1-methylpiperidin-4-ylmethoxy)qu--inazoline (ZD6474; Example 2 within WO 01/32651),4-(4-fluoro-2-methylindol-5-yloxy)-6-methoxy-7-(3-pyrrolidin-1-ylpropoxy)--quinazoline (AZD2171; Example 240 within WO 00/47212), vatalanib(PTK787; WO 98/35985) and SU11248 (sunitinib; WO 01/60814), andcompounds such as those disclosed in PCT Publication Nos. WO 97/22596,WO 97/30035, WO 97/32856, and WO 98/13354).

In certain embodiments, the payload is an antibody or an antibodyfragment. In certain embodiments, the payload antibody or fragment canbe encoded by any of the immunoglobulin genes recognized by those ofskill in the art. The immunoglobulin genes include, but are not limitedto, the κ, λ, α, γ (IgG1, IgG2, IgG3, and IgG4), δ, ε and μ constantregion genes, as well as the immunoglobulin variable region genes. Theterm includes full-length antibodies and antibody fragments recognizedby those of skill in the art, and variants thereof. Exemplary fragmentsinclude but are not limited to Fv, Fc, Fab, and (Fab′)₂, single chain Fv(scFv), diabodies, triabodies, tetrabodies, bifunctional hybridantibodies, CDR1, CDR2, CDR3, combinations of CDR's, variable regions,framework regions, constant regions, and the like.

In certain embodiments, the payload can be one or more water-solublepolymers. A wide variety of macromolecular polymers and other moleculescan be linked to antigen-binding polypeptides of the present inventionto modulate biological properties of the antibody, and/or provide newbiological properties to the antibody. These macromolecular polymers canbe linked to the antibody via a naturally encoded amino acid, via anon-naturally encoded amino acid, or any functional substituent of anatural or non-natural amino acid, or any substituent or functionalgroup added to a natural or non-natural amino acid. The molecular weightof the polymer may be of a wide range, including but not limited to,between about 100 Da and about 100,000 Da or more.

The polymer selected may be water soluble so that the protein to whichit is attached does not precipitate in an aqueous environment, such as aphysiological environment. The polymer may be branched or unbranched.Preferably, for therapeutic use of the end-product preparation, thepolymer will be pharmaceutically acceptable.

The proportion of polyethylene glycol molecules to antibody moleculeswill vary, as will their concentrations in the reaction mixture. Ingeneral, the optimum ratio (in terms of efficiency of reaction in thatthere is minimal excess unreacted protein or polymer) may be determinedby the molecular weight of the polyethylene glycol selected and on thenumber of available reactive groups available. As relates to molecularweight, typically the higher the molecular weight of the polymer, thefewer number of polymer molecules which may be attached to the protein.Similarly, branching of the polymer should be taken into account whenoptimizing these parameters. Generally, the higher the molecular weight(or the more branches) the higher the polymer:protein ratio.

The water soluble polymer may be any structural form including but notlimited to linear, forked or branched. Typically, the water-solublepolymer is a poly(alkylene glycol), such as poly(ethylene glycol) (PEG),but other water-soluble polymers can also be employed. By way ofexample, PEG is used to describe certain embodiments of this invention.

PEG is a well-known, water soluble polymer that is commerciallyavailable or can be prepared by ring-opening polymerization of ethyleneglycol according to methods well known in the art (Sandler and Karo,Polymer Synthesis, Academic Press, New York, Vol. 3, pages 138-161). Theterm “PEG” is used broadly to encompass any polyethylene glycolmolecule, without regard to size or to modification at an end of thePEG, and can be represented as linked to the antibody by the formula:XO—(CH₂CH₂O)═_(n)—CH₂CH₂—Y where n is 2 to 10,000 and X is H or aterminal modification, including but not limited to, a C₁₋₄ alkyl.

In some cases, a PEG used in the invention terminates on one end withhydroxy or methoxy, i.e., X is H or CH₃ (“methoxy PEG”). Alternatively,the PEG can terminate with a reactive group, thereby forming abifunctional polymer. Typical reactive groups can include those reactivegroups that are commonly used to react with the functional groups foundin the common amino acids (including but not limited to, maleimidegroups, activated carbonates (including but not limited to,p-nitrophenyl ester), activated esters (including but not limited to,N-hydroxysuccinimide, p-nitrophenyl ester) and aldehydes) as well asfunctional groups that are inert to the 20 common amino acids but thatreact specifically with complementary functional groups present innon-naturally encoded amino acids (including but not limited to, azidegroups, alkyne groups). It is noted that the other end of the PEG, whichis shown in the above formula by Y, will attach either directly orindirectly to an antigen-binding polypeptide via a naturally-occurringor non-naturally encoded amino acid. For instance, Y may be an amide,carbamate or urea linkage to an amine group (including but not limitedto, the epsilon amine of lysine or the N-terminus) of the polypeptide.Alternatively, Y may be a maleimide linkage to a thiol group (includingbut not limited to, the thiol group of cysteine). Alternatively, Y maybe a linkage to a residue not commonly accessible via the 20 commonamino acids. For example, an azide group on the PEG can be reacted withan alkyne group on the antibody to form a Huisgen [3+2] cycloadditionproduct. Alternatively, an alkyne group on the PEG can be reacted withan azide group present in a non-naturally encoded amino acid to form asimilar product. In some embodiments, a strong nucleophile (includingbut not limited to, hydrazine, hydrazide, hydroxylamine, semicarbazide)can be reacted with an aldehyde or ketone group present in anon-naturally encoded amino acid to form a hydrazone, oxime orsemicarbazone, as applicable, which in some cases can be further reducedby treatment with an appropriate reducing agent. Alternatively, thestrong nucleophile can be incorporated into the antibody via anon-naturally encoded amino acid and used to react preferentially with aketone or aldehyde group present in the water soluble polymer.

Any molecular mass for a PEG can be used as practically desired,including but not limited to, from about 100 Daltons (Da) to 100,000 Daor more as desired (including but not limited to, sometimes 0.1-50 kDaor 10-40 kDa). Branched chain PEGs, including but not limited to, PEGmolecules with each chain having a MW ranging from 1-100 kDa (includingbut not limited to, 1-50 kDa or 5-20 kDa) can also be used. A wide rangeof PEG molecules are described in, including but not limited to, theShearwater Polymers, Inc. catalog, Nektar Therapeutics catalog,incorporated herein by reference.

Generally, at least one terminus of the PEG molecule is available forreaction with the non-naturally-encoded amino acid. For example, PEGderivatives bearing alkyne and azide moieties for reaction with aminoacid side chains can be used to attach PEG to non-naturally encodedamino acids as described herein. If the non-naturally encoded amino acidcomprises an azide, then the PEG will typically contain either an alkynemoiety to effect formation of the [3+2] cycloaddition product or anactivated PEG species (i.e., ester, carbonate) containing a phosphinegroup to effect formation of the amide linkage. Alternatively, if thenon-naturally encoded amino acid comprises an alkyne, then the PEG willtypically contain an azide moiety to effect formation of the [3+2]Huisgen cycloaddition product. If the non-naturally encoded amino acidcomprises a carbonyl group, the PEG will typically comprise a potentnucleophile (including but not limited to, a hydrazide, hydrazine,hydroxylamine, or semicarbazide functionality) in order to effectformation of corresponding hydrazone, oxime, and semicarbazone linkages,respectively. In other alternatives, a reverse of the orientation of thereactive groups described above can be used, i.e., an azide moiety inthe non-naturally encoded amino acid can be reacted with a PEGderivative containing an alkyne.

In some embodiments, the Fc protein variant with a PEG derivativecontains a chemical functionality that is reactive with the chemicalfunctionality present on the side chain of the non-naturally encodedamino acid.

In certain embodiments, the payload is an azide- or acetylene-containingpolymer comprising a water soluble polymer backbone having an averagemolecular weight from about 800 Da to about 100,000 Da. The polymerbackbone of the water-soluble polymer can be poly(ethylene glycol).However, it should be understood that a wide variety of water solublepolymers including but not limited to poly(ethylene)glycol and otherrelated polymers, including poly(dextran) and poly(propylene glycol),are also suitable for use in the practice of this invention and that theuse of the term PEG or poly(ethylene glycol) is intended to encompassand include all such molecules. The term PEG includes, but is notlimited to, poly(ethylene glycol) in any of its forms, includingbifunctional PEG, multiarmed PEG, derivatized PEG, forked PEG, branchedPEG, pendent PEG (i.e. PEG or related polymers having one or morefunctional groups pendent to the polymer backbone), or PEG withdegradable linkages therein.

The polymer backbone can be linear or branched. Branched polymerbackbones are generally known in the art. Typically, a branched polymerhas a central branch core moiety and a plurality of linear polymerchains linked to the central branch core. PEG is commonly used inbranched forms that can be prepared by addition of ethylene oxide tovarious polyols, such as glycerol, glycerol oligomers, pentaerythritoland sorbitol. The central branch moiety can also be derived from severalamino acids, such as lysine. The branched poly(ethylene glycol) can berepresented in general form as R(-PEG-OH)_(m) in which R is derived froma core moiety, such as glycerol, glycerol oligomers, or pentaerythritol,and m represents the number of arms. Multi-armed PEG molecules, such asthose described in U.S. Pat. Nos. 5,932,462 5,643,575; 5,229,490;4,289,872; U.S. Pat. Appl. 2003/0143596; WO 96/21469; and WO 93/21259,each of which is incorporated by reference herein in its entirety, canalso be used as the polymer backbone.

Branched PEG can also be in the form of a forked PEG represented byPEG(-YCHZ₂)_(n), where Y is a linking group and Z is an activatedterminal group linked to CH by a chain of atoms of defined length.

Yet another branched form, the pendant PEG, has reactive groups, such ascarboxyl, along the PEG backbone rather than at the end of PEG chains.

In addition to these forms of PEG, the polymer can also be prepared withweak or degradable linkages in the backbone. For example, PEG can beprepared with ester linkages in the polymer backbone that are subject tohydrolysis. As shown below, this hydrolysis results in cleavage of thepolymer into fragments of lower molecular weight:-PEG-CO₂-PEG-+H₂O→PEG-CO₂H+HO-PEG- It is understood by those skilled inthe art that the term poly(ethylene glycol) or PEG represents orincludes all the forms known in the art including but not limited tothose disclosed herein.

Many other polymers are also suitable for use in the present invention.In some embodiments, polymer backbones that are water-soluble, with from2 to about 300 termini, are particularly useful in the invention.Examples of suitable polymers include, but are not limited to, otherpoly(alkylene glycols), such as poly(propylene glycol) (“PPG”),copolymers thereof (including but not limited to copolymers of ethyleneglycol and propylene glycol), terpolymers thereof, mixtures thereof, andthe like. Although the molecular weight of each chain of the polymerbackbone can vary, it is typically in the range of from about 800 Da toabout 100,000 Da, often from about 6,000 Da to about 80,000 Da.

Those of ordinary skill in the art will recognize that the foregoinglist for substantially water soluble backbones is by no means exhaustiveand is merely illustrative, and that all polymeric materials having thequalities described above are contemplated as being suitable for use inthe present invention.

In some embodiments of the present invention the polymer derivatives are“multi-functional”, meaning that the polymer backbone has at least twotermini, and possibly as many as about 300 termini, functionalized oractivated with a functional group. Multifunctional polymer derivativesinclude, but are not limited to, linear polymers having two termini,each terminus being bonded to a functional group which may be the sameor different.

In one embodiment, the polymer derivative has the structure:X-A-POLY-B-N═N═N wherein: N═N═N is an azide moiety; B is a linkingmoiety, which may be present or absent; POLY is a water-solublenon-antigenic polymer; A is a linking moiety, which may be present orabsent and which may be the same as B or different; and X is a secondfunctional group. Examples of a linking moiety for A and B include, butare not limited to, a multiply-functionalized alkyl group containing upto 18, and more preferably between 1-10 carbon atoms. A heteroatom suchas nitrogen, oxygen or sulfur may be included with the alkyl chain. Thealkyl chain may also be branched at a heteroatom. Other examples of alinking moiety for A and B include, but are not limited to, a multiplyfunctionalized aryl group, containing up to 10 and more preferably 5-6carbon atoms. The aryl group may be substituted with one more carbonatoms, nitrogen, oxygen or sulfur atoms. Other examples of suitablelinking groups include those linking groups described in U.S. Pat. Nos.5,932,462; 5,643,575; and U.S. Pat. Appl. Publication 2003/0143596, eachof which is incorporated by reference herein. Those of ordinary skill inthe art will recognize that the foregoing list for linking moieties isby no means exhaustive and is merely illustrative, and that all linkingmoieties having the qualities described above are contemplated to besuitable for use in the present invention.

Examples of suitable functional groups for use as X include, but are notlimited to, hydroxyl, protected hydroxyl, alkoxyl, active ester, such asN-hydroxysuccinimidyl esters and 1-benzotriazolyl esters, activecarbonate, such as N-hydroxysuccinimidyl carbonates and 1-benzotriazolylcarbonates, acetal, aldehyde, aldehyde hydrates, alkenyl, acrylate,methacrylate, acrylamide, active sulfone, amine, aminooxy, protectedamine, hydrazide, protected hydrazide, protected thiol, carboxylic acid,protected carboxylic acid, isocyanate, isothiocyanate, maleimide,vinylsulfone, dithiopyridine, vinylpyridine, iodoacetamide, epoxide,glyoxals, diones, mesylates, tosylates, tresylate, alkene, ketone, andazide. As is understood by those skilled in the art, the selected Xmoiety should be compatible with the azide group so that reaction withthe azide group does not occur. The azide-containing polymer derivativesmay be homobifunctional, meaning that the second functional group (i.e.,X) is also an azide moiety, or heterobifunctional, meaning that thesecond functional group is a different functional group.

The term “protected” refers to the presence of a protecting group ormoiety that prevents reaction of the chemically reactive functionalgroup under certain reaction conditions. The protecting group will varydepending on the type of chemically reactive group being protected. Forexample, if the chemically reactive group is an amine or a hydrazide,the protecting group can be selected from the group oftert-butyloxycarbonyl (t-Boc) and 9-fluorenylmethoxycarbonyl (Fmoc). Ifthe chemically reactive group is a thiol, the protecting group can beorthopyridyldisulfide. If the chemically reactive group is a carboxylicacid, such as butanoic or propionic acid, or a hydroxyl group, theprotecting group can be benzyl or an alkyl group such as methyl, ethyl,or tert-butyl. Other protecting groups known in the art may also be usedin the present invention.

Specific examples of terminal functional groups in the literatureinclude, but are not limited to, N-succinimidyl carbonate (see e.g.,U.S. Pat. Nos. 5,281,698, 5,468,478), amine (see, e.g., Buckmann et al.Makromol. Chem. 182:1379 (1981), Zaplipsky et al. Eur. Polym. J. 19:1177(1983)), hydrazide (See, e.g., Andresz et al. Makromol. Chem. 179:301(1978)), succinimidyl propionate and succinimidyl butanoate (see, e.g.,Olson et al. in Poly(ethylene glycol) Chemistry & BiologicalApplications, pp 170-181, Harris & Zaplipsky Eds., ACS, Washington,D.C., 1997; see also U.S. Pat. No. 5,672,662), succinimidyl succinate(See, e.g., Abuchowski et al. Cancer Biochem. Biophys. 7:175 (1984) andJoppich et al. Macrolol. Chem. 180:1381 (1979), succinimidyl ester (see,e.g., U.S. Pat. No. 4,670,417), benzotriazole carbonate (see, e.g., U.S.Pat. No. 5,650,234), glycidyl ether (see, e.g., Pitha et al. Eur. JBiochem. 94:11 (1979), Elling et al., Biotech. Appl. Biochem. 13:354(1991), oxycarbonylimidazole (see, e.g., Beauchamp, et al., Anal.Biochem. 131:25 (1983), Tondelli et al. J. Controlled Release 1:251(1985)), p-nitrophenyl carbonate (see, e.g., Veronese, et al., Appl.Biochem. Biotech., 11: 141 (1985); and Sartore et al., Appl. Biochem.Biotech., 27:45 (1991)), aldehyde (see, e.g., Harris et al. J. Polym.Sci. Chem. Ed. 22:341 (1984), U.S. Pat. No. 5,824,784, U.S. Pat. No.5,252,714), maleimide (see, e.g., Goodson et al. Bio/Technology 8:343(1990), Romani et al. in Chemistry of Peptides and Proteins 2:29(1984)), and Kogan, Synthetic Comm. 22:2417 (1992)),orthopyridyl-disulfide (see, e.g., Woghiren, et al. Bioconj. Chem. 4:314(1993)), acrylol (see, e.g., Sawhney et al., Macromolecules, 26:581(1993)), vinylsulfone (see, e.g., U.S. Pat. No. 5,900,461). All of theabove references and patents are incorporated herein by reference.

In certain embodiments of the present invention, the polymer derivativesof the invention comprise a polymer backbone having the structure:X—CH₂CH₂O—(CH₂CH₂O)_(n)—CH₂CH₂—N═N═N wherein: X is a functional group asdescribed above; and n is about 20 to about 4000. In another embodiment,the polymer derivatives of the invention comprise a polymer backbonehaving the structure:X—CH₂CH₂O—(CH₂CH₂O)_(n)—CH₂CH₂—O—(CH—₂)_(m)—W—N═N═N wherein: W is analiphatic or aromatic linker moiety comprising between 1-10 carbonatoms; n is about 20 to about 4000; X is a functional group as describedabove; and m is between 1 and 10.

The azide-containing PEG derivatives of the invention can be prepared bya variety of methods known in the art and/or disclosed herein. In onemethod, shown below, a water soluble polymer backbone having an averagemolecular weight from about 800 Da to about 100,000 Da, the polymerbackbone having a first terminus bonded to a first functional group anda second terminus bonded to a suitable leaving group, is reacted with anazide anion (which may be paired with any of a number of suitablecounter-ions, including sodium, potassium, tert-butylammonium and soforth). The leaving group undergoes a nucleophilic displacement and isreplaced by the azide moiety, affording the desired azide-containing PEGpolymer as shown in the following: X-PEG-L+N₃ ⁻→X-PEG-N₃.

As shown, a suitable polymer backbone for use in the present inventionhas the formula X-PEG-L, wherein PEG is poly(ethylene glycol) and X is afunctional group that does not react with azide groups and L is asuitable leaving group. Examples of suitable functional groups include,but are not limited to, hydroxyl, protected hydroxyl, acetal, alkenyl,amine, aminooxy, protected amine, protected hydrazide, protected thiol,carboxylic acid, protected carboxylic acid, maleimide, dithiopyridine,and vinylpyridine, and ketone. Examples of suitable leaving groupsinclude, but are not limited to, chloride, bromide, iodide, mesylate,tresylate, and tosylate.

In another method for preparation of the azide-containing polymerderivatives of the present invention, a linking agent bearing an azidefunctionality is contacted with a water soluble polymer backbone havingan average molecular weight from about 800 Da to about 100,000 Da,wherein the linking agent bears a chemical functionality that will reactselectively with a chemical functionality on the PEG polymer, to form anazide-containing polymer derivative product wherein the azide isseparated from the polymer backbone by a linking group.

An exemplary reaction scheme is shown below:X-PEG-M+N-linker-N═N═N→PG-X-PEG-linker-N═N═N wherein: PEG ispoly(ethylene glycol) and X is a capping group such as alkoxy or afunctional group as described above; and M is a functional group that isnot reactive with the azide functionality but that will reactefficiently and selectively with the N functional group.

Examples of suitable functional groups include, but are not limited to,M being a carboxylic acid, carbonate or active ester if N is an amine; Mbeing a ketone if N is a hydrazide or aminooxy moiety; M being a leavinggroup if N is a nucleophile.

Purification of the crude product may be accomplished by known methodsincluding, but are not limited to, precipitation of the product followedby chromatography, if necessary.

A more specific example is shown below in the case of PEG diamine, inwhich one of the amines is protected by a protecting group moiety suchas tert-butyl-Boc and the resulting mono-protected PEG diamine isreacted with a linking moiety that bears the azide functionality:BocHN-PEG-NH₂+HO₂C—(CH₂)₃—N═N═N.

In this instance, the amine group can be coupled to the carboxylic acidgroup using a variety of activating agents such as thionyl chloride orcarbodiimide reagents and N-hydroxysuccinimide or N-hydroxybenzotriazoleto create an amide bond between the monoamine PEG derivative and theazide-bearing linker moiety. After successful formation of the amidebond, the resulting N-tert-butyl-Boc-protected azide-containingderivative can be used directly to modify bioactive molecules or it canbe further elaborated to install other useful functional groups. Forinstance, the N-t-Boc group can be hydrolyzed by treatment with strongacid to generate an omega-amino-PEG-azide. The resulting amine can beused as a synthetic handle to install other useful functionality such asmaleimide groups, activated disulfides, activated esters and so forthfor the creation of valuable heterobifunctional reagents.

Heterobifunctional derivatives are particularly useful when it isdesired to attach different molecules to each terminus of the polymer.For example, the omega-N-amino-N-azido PEG would allow the attachment ofa molecule having an activated electrophilic group, such as an aldehyde,ketone, activated ester, activated carbonate and so forth, to oneterminus of the PEG and a molecule having an acetylene group to theother terminus of the PEG.

In another embodiment of the invention, the polymer derivative has thestructure: X-A-POLY-B-C═C—R wherein: R can be either H or an alkyl,alkene, alkyoxy, or aryl or substituted aryl group; B is a linkingmoiety, which may be present or absent; POLY is a water-solublenon-antigenic polymer; A is a linking moiety, which may be present orabsent and which may be the same as B or different; and X is a secondfunctional group.

Examples of a linking moiety for A and B include, but are not limitedto, a multiply-functionalized alkyl group containing up to 18, and morepreferably between 1-10 carbon atoms. A heteroatom such as nitrogen,oxygen or sulfur may be included with the alkyl chain. The alkyl chainmay also be branched at a heteroatom. Other examples of a linking moietyfor A and B include, but are not limited to, a multiply functionalizedaryl group, containing up to 10 and more preferably 5-6 carbon atoms.The aryl group may be substituted with one more carbon atoms, nitrogen,oxygen, or sulfur atoms. Other examples of suitable linking groupsinclude those linking groups described in U.S. Pat. Nos. 5,932,462 and5,643,575 and U.S. Pat. Appl. Publication 2003/0143596, each of which isincorporated by reference herein. Those of ordinary skill in the artwill recognize that the foregoing list for linking moieties is by nomeans exhaustive and is intended to be merely illustrative, and that awide variety of linking moieties having the qualities described aboveare contemplated to be useful in the present invention.

Examples of suitable functional groups for use as X include hydroxyl,protected hydroxyl, alkoxyl, active ester, such as N-hydroxysuccinimidylesters and 1-benzotriazolyl esters, active carbonate, such asN-hydroxysuccinimidyl carbonates and 1-benzotriazolyl carbonates,acetal, aldehyde, aldehyde hydrates, alkenyl, acrylate, methacrylate,acrylamide, active sulfone, amine, aminooxy, protected amine, hydrazide,protected hydrazide, protected thiol, carboxylic acid, protectedcarboxylic acid, isocyanate, isothiocyanate, maleimide, vinylsulfone,dithiopyridine, vinylpyridine, iodoacetamide, epoxide, glyoxals, diones,mesylates, tosylates, and tresylate, alkene, ketone, and acetylene. Aswould be understood, the selected X moiety should be compatible with theacetylene group so that reaction with the acetylene group does notoccur. The acetylene-containing polymer derivatives may behomobifunctional, meaning that the second functional group (i.e., X) isalso an acetylene moiety, or heterobifunctional, meaning that the secondfunctional group is a different functional group.

In another embodiment of the present invention, the polymer derivativescomprise a polymer backbone having the structure:X—CH₂CH₂O—(CH₂CH₂O)_(n)—CH₂CH₂—O—(CH₂)_(m)—C═CH wherein: X is afunctional group as described above; n is about 20 to about 4000; and mis between 1 and 10. Specific examples of each of the heterobifunctionalPEG polymers are shown below.

The acetylene-containing PEG derivatives of the invention can beprepared using methods known to those skilled in the art and/ordisclosed herein. In one method, a water soluble polymer backbone havingan average molecular weight from about 800 Da to about 100,000 Da, thepolymer backbone having a first terminus bonded to a first functionalgroup and a second terminus bonded to a suitable nucleophilic group, isreacted with a compound that bears both an acetylene functionality and aleaving group that is suitable for reaction with the nucleophilic groupon the PEG. When the PEG polymer bearing the nucleophilic moiety and themolecule bearing the leaving group are combined, the leaving groupundergoes a nucleophilic displacement and is replaced by thenucleophilic moiety, affording the desired acetylene-containing polymer:X-PEG-Nu+L-A-C→X-PEG-Nu-A-C═CR′.

As shown, a preferred polymer backbone for use in the reaction has theformula X-PEG-Nu, wherein PEG is poly(ethylene glycol), Nu is anucleophilic moiety and X is a functional group that does not react withNu, L or the acetylene functionality.

Examples of Nu include, but are not limited to, amine, alkoxy, aryloxy,sulfhydryl, imino, carboxylate, hydrazide, aminoxy groups that wouldreact primarily via a SN2-type mechanism. Additional examples of Nugroups include those functional groups that would react primarily via anucleophilic addition reaction. Examples of L groups include chloride,bromide, iodide, mesylate, tresylate, and tosylate and other groupsexpected to undergo nucleophilic displacement as well as ketones,aldehydes, thioesters, olefins, alpha-beta unsaturated carbonyl groups,carbonates and other electrophilic groups expected to undergo additionby nucleophiles.

In another embodiment of the present invention, A is an aliphatic linkerof between 1-10 carbon atoms or a substituted aryl ring of between 6-14carbon atoms. X is a functional group which does not react with azidegroups and L is a suitable leaving group.

In another method for preparation of the acetylene-containing polymerderivatives of the invention, a PEG polymer having an average molecularweight from about 800 Da to about 100,000 Da, bearing either a protectedfunctional group or a capping agent at one terminus and a suitableleaving group at the other terminus is contacted by an acetylene anion.

Water soluble polymers can be linked to the Fc proteins of theinvention. The water soluble polymers may be linked via a non-naturallyencoded amino acid incorporated in the Fc proteins or any functionalgroup or substituent of a non-naturally encoded or naturally encodedamino acid, or any functional group or substituent added to anon-naturally encoded or naturally encoded amino acid. Alternatively,the water soluble polymers are linked to an antigen-binding polypeptideincorporating a non-naturally encoded amino acid via anaturally-occurring amino acid (including but not limited to, cysteine,lysine or the amine group of the N-terminal residue). In some cases, theFc proteins of the invention comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, 10non-natural amino acids, wherein one or more non-naturally-encoded aminoacid(s) are linked to water soluble polymer(s) (including but notlimited to, PEG and/or oligosaccharides). In some cases, the Fc proteinsof the invention further comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or morenaturally-encoded amino acid(s) linked to water soluble polymers. Insome cases, the Fc protein of the invention comprise one or morenon-naturally encoded amino acid(s) linked to water soluble polymers andone or more naturally-occurring amino acids linked to water solublepolymers. In some embodiments, the water soluble polymers used in thepresent invention enhance the serum half-life of the Fc proteinsrelative to the unconjugated form.

The number of water soluble polymers linked to an antigen-bindingpolypeptide (i.e., the extent of PEGylation or glycosylation) of thepresent invention can be adjusted to provide an altered (including butnot limited to, increased or decreased) pharmacologic, pharmacokineticor pharmacodynamic characteristic such as in vivo half-life. In someembodiments, the half-life of Fc protein is increased at least about 10,20, 30, 40, 50, 60, 70, 80, 90 percent, 2-fold, 5-fold, 10-fold,50-fold, or at least about 100-fold over an unmodified polypeptide.

In one embodiment of the present invention, an antigen-bindingpolypeptide comprising a carbonyl-containing non-naturally encoded aminoacid is modified with a PEG derivative that contains a terminalhydrazine, hydroxylamine, hydrazide or semicarbazide moiety that islinked directly to the PEG backbone.

In some embodiments, the hydroxylamine-terminal PEG derivative will havethe structure: RO—(CH₂CH₂O)_(n)—O—(CH₂)_(m)—O—NH₂ where R is a simplealkyl (methyl, ethyl, propyl, etc.), m is 2-10 and n is 100-1,000 (i.e.,average molecular weight is between 5-40 kDa).

In some embodiments, the hydrazine- or hydrazide-containing PEGderivative will have the structure:RO—(CH₂CH₂O)_(n)—O—(CH₂)_(m)—X—NH—NH₂ where R is a simple alkyl (methyl,ethyl, propyl, etc.), m is 2-10 and n is 100-1,000 and X is optionally acarbonyl group (C═O) that can be present or absent.

In some embodiments, the semicarbazide-containing PEG derivative willhave the structure: RO—(CH₂CH₂O)_(n)—O—(CH₂)_(m)—NH—C(O)—NH—NH₂ where Ris a simple alkyl (methyl, ethyl, propyl, etc.), m is 2-10 and n is100-1,000.

In another embodiment of the invention, an antigen-binding polypeptidecomprising a carbonyl-containing amino acid is modified with a PEGderivative that contains a terminal hydroxylamine, hydrazide, hydrazine,or semicarbazide moiety that is linked to the PEG backbone by means ofan amide linkage.

In some embodiments, the hydroxylamine-terminal PEG derivatives have thestructure: RO—(CH₂CH₂O)_(n)—O—(CH₂)₂—NH—C(O)(CH₂)_(m)—O—NH₂ where R is asimple alkyl (methyl, ethyl, propyl, etc.), m is 2-10 and n is 100-1,000(i.e., average molecular weight is between 5-40 kDa).

In some embodiments, the hydrazine- or hydrazide-containing PEGderivatives have the structure:RO—(CH₂CH₂O)_(n)—O—(CH₂)₂—NH—C(O)(CH₂)_(m)—X—NH—NH₂ where R is a simplealkyl (methyl, ethyl, propyl, etc.), m is 2-10, n is 100-1,000 and X isoptionally a carbonyl group (C═O) that can be present or absent.

In some embodiments, the semicarbazide-containing PEG derivatives havethe structure: RO—(CH₂CH₂O)_(n)—O—(CH₂)₂—NH—C(O)(CH₂)_(m)—NH—C(O)—NH—NH₂where R is a simple alkyl (methyl, ethyl, propyl, etc.), m is 2-10 and nis 100-1,000.

In another embodiment of the invention, an Fc protein comprising acarbonyl-containing amino acid is modified with a branched PEGderivative that contains a terminal hydrazine, hydroxylamine, hydrazideor semicarbazide moiety, with each chain of the branched PEG having a MWranging from 10-40 kDa and, more preferably, from 5-20 kDa.

In another embodiment of the invention, an Fc protein comprising anon-naturally encoded amino acid is modified with a PEG derivativehaving a branched structure. For instance, in some embodiments, thehydrazine- or hydrazide-terminal PEG derivative will have the followingstructure: [RO—(CH₂CH₂O)—O—(CH₂)₂—NH—C(O)]₂CH(CH—₂)_(m)—X—NH—NH₂ where Ris a simple alkyl (methyl, ethyl, propyl, etc.), m is 2-10 and n is100-1,000, and X is optionally a carbonyl group (C═O) that can bepresent or absent.

In some embodiments, the PEG derivatives containing a semicarbazidegroup will have the structure:[RO—(CH₂CH₂O)_(n)—O—(CH₂)₂—C(O)—NH—CH₂—CH₂]₂CH—X—(CH₂)_(m)—NH—C(O)—NH—NH₂where R is a simple alkyl (methyl, ethyl, propyl, etc.), X is optionallyNH, O, S, C(O) or not present, m is 2-10 and n is 100-1,000.

In some embodiments, the PEG derivatives containing a hydroxylaminegroup will have the structure:[RO—(CH₂CH₂O)_(n)—O—(CH₂)₂—C(O)—NH—CH₂—CH₂]₂CH—X—(CH₂)_(m)—O—NH₂ where Ris a simple alkyl (methyl, ethyl, propyl, etc.), X is optionally NH, O,S, C(O) or not present, m is 2-10 and n is 100-1,000.

The degree and sites at which the water soluble polymer(s) are linked tothe Fc proteins can modulate the binding of the Fc proteins to anantigen or receptor.

Methods and chemistry for activation of polymers as well as forconjugation of peptides are described in the literature and are known inthe art. Commonly used methods for activation of polymers include, butare not limited to, activation of functional groups with cyanogenbromide, periodate, glutaraldehyde, biepoxides, epichlorohydrin,divinylsulfone, carbodiimide, sulfonyl halides, trichlorotriazine, etc.(see, R. F. Taylor, (1991), PROTEIN IMMOBILISATION. FUNDAMENTAL ANDAPPLICATIONS, Marcel Dekker, N.Y.; S. S. Wong, (1992), CHEMISTRY OFPROTEIN CONJUGATION AND CROSSLINKING, CRC Press, Boca Raton; G. T.Hermanson et al., (1993), IMMOBILIZED AFFINITY LIGAND TECHNIQUES,Academic Press, N.Y.; Dunn, R. L., et al., Eds. POLYMERIC DRUGS AND DRUGDELIVERY SYSTEMS, ACS Symposium Series Vol. 469, American ChemicalSociety, Washington, D.C. 1991).

Several reviews and monographs on the functionalization and conjugationof PEG are available. See, for example, Harris, Macronol. Chem. Phys.C25: 325-373 (1985); Scouten, Methods in Enzymology 135: 30-65 (1987);Wong et al., Enzyme Microb. Technol. 14: 866-874 (1992); Delgado et al.,Critical Reviews in Therapeutic Drug Carrier Systems 9: 249-304 (1992);Zalipsky, Bioconjugate Chem. 6: 150-165 (1995).

Methods for activation of polymers can also be found in WO 94/17039,U.S. Pat. No. 5,324,844, WO 94/18247, WO 94/04193, U.S. Pat. No.5,219,564, U.S. Pat. No. 5,122,614, WO 90/13540, U.S. Pat. No.5,281,698, and WO 93/15189, and for conjugation between activatedpolymers and enzymes including but not limited to Coagulation FactorVIII (WO 94/15625), hemoglobin (WO 94/09027), oxygen carrying molecule(U.S. Pat. No. 4,412,989), ribonuclease and superoxide dismutase(Veronese at al., App. Biochem. Biotech. 11: 141-45 (1985)). Allreferences and patents cited are incorporated by reference herein.

PEGylation (i.e., addition of any water soluble polymer) ofantigen-binding polypeptides containing a non-naturally encoded aminoacid, such as p-azido-L-phenylalanine, is carried out by any convenientmethod. For example, Fc protein is PEGylated with an alkyne-terminatedmPEG derivative. Briefly, an excess of solid mPEG(5000)-O—CH₂—C═CH isadded, with stirring, to an aqueous solution of p-azido-L-Phe-containingFc protein at room temperature. Typically, the aqueous solution isbuffered with a buffer having a pK_(a) near the pH at which the reactionis to be carried out (generally about pH 4-10). Examples of suitablebuffers for PEGylation at pH 7.5, for instance, include, but are notlimited to, HEPES, phosphate, borate, TRIS-HCl, EPPS, and TES. The pH iscontinuously monitored and adjusted if necessary. The reaction istypically allowed to continue for between about 1-48 hours.

The reaction products are subsequently subjected to hydrophobicinteraction chromatography to separate the PEGylated Fc protein variantsfrom free mPEG(5000)-O—CH₂—C═CH and any high-molecular weight complexesof the pegylated Fc protein which may form when unblocked PEG isactivated at both ends of the molecule, thereby crosslinking Fc proteinvariant molecules. The conditions during hydrophobic interactionchromatography are such that free mPEG(5000)-O—CH₂—C═CH flows throughthe column, while any crosslinked PEGylated Fc protein variant complexeselute after the desired forms, which contain one Fc protein variantmolecule conjugated to one or more PEG groups. Suitable conditions varydepending on the relative sizes of the cross-linked complexes versus thedesired conjugates and are readily determined by those skilled in theart. The eluent containing the desired conjugates is concentrated byultrafiltration and desalted by diafiltration.

If necessary, the PEGylated Fc proteins obtained from the hydrophobicchromatography can be purified further by one or more procedures knownto those skilled in the art including, but are not limited to, affinitychromatography; anion- or cation-exchange chromatography (using,including but not limited to, DEAE SEPHAROSE); chromatography on silica;reverse phase HPLC; gel filtration (using, including but not limited to,SEPHADEX G-75); hydrophobic interaction chromatography; size-exclusionchromatography, metal-chelate chromatography;ultrafiltration/diafiltration; ethanol precipitation; ammonium sulfateprecipitation; chromatofocusing; displacement chromatography;electrophoretic procedures (including but not limited to preparativeisoelectric focusing), differential solubility (including but notlimited to ammonium sulfate precipitation), or extraction. Apparentmolecular weight may be estimated by GPC by comparison to globularprotein standards (PROTEIN PURIFICATION METHODS, A PRACTICAL APPROACH(Harris & Angal, Eds.) IRL Press 1989, 293-306). The purity of the Fcprotein-PEG conjugate can be assessed by proteolytic degradation(including but not limited to, trypsin cleavage) followed by massspectrometry analysis. Pepinsky B., et al., J. Pharmcol. & Exp. Ther.297(3):1059-66 (2001).

A water soluble polymer linked to an amino acid of an Fc protein of theinvention can be further derivatized or substituted without limitation.

In another embodiment of the invention, an antigen-binding polypeptideis modified with a PEG derivative that contains an azide moiety thatwill react with an alkyne moiety present on the side chain of thenon-naturally encoded amino acid. In general, the PEG derivatives willhave an average molecular weight ranging from 1-100 kDa and, in someembodiments, from 10-40 kDa.

In some embodiments, the azide-terminal PEG derivative will have thestructure: RO—(CH₂CH₂O)_(n)—O—(CH₂)_(m)—N₃ where R is a simple alkyl(methyl, ethyl, propyl, etc.), m is 2-10 and n is 100-1,000 (i.e.,average molecular weight is between 5-40 kDa).

In another embodiment, the azide-terminal PEG derivative will have thestructure: RO—(CH₂CH₂O)_(n)—O—(CH₂)_(m)—NH—C(O)—(CH₂)_(p)—N₃, where R isa simple alkyl (methyl, ethyl, propyl, etc.), m is 2-10, p is 2-10 and nis 100-1,000 (i.e., average molecular weight is between 5-40 kDa).

In another embodiment of the invention, an Fc protein comprising aalkyne-containing amino acid is modified with a branched PEG derivativethat contains a terminal azide moiety, with each chain of the branchedPEG having a MW ranging from 10-40 kDa and, more preferably, from 5-20kDa. For instance, in some embodiments, the azide-terminal PEGderivative will have the following structure:[RO—(CH₂CH₂O)_(n)—O—(CH₂)₂—NH—C(O)]₂CH(CH—₂)_(m)—X—(CH₂)_(p)—N₃ where Ris a simple alkyl (methyl, ethyl, propyl, etc.), m is 2-10, p is 2-10,and n is 100-1,000, and X is optionally an O, N, S or carbonyl group(C═O), in each case that can be present or absent.

In another embodiment of the invention, an antigen-binding polypeptideis modified with a PEG derivative that contains an alkyne moiety thatwill react with an azide moiety present on the side chain of thenon-naturally encoded amino acid.

In some embodiments, the alkyne-terminal PEG derivative will have thefollowing structure: RO—(CH₂CH₂O)_(n)—O—(CH₂)_(m)—C═CH where R is asimple alkyl (methyl, ethyl, propyl, etc.), m is 2-10 and n is 100-1,000(i.e., average molecular weight is between 5-40 kDa).

In another embodiment of the invention, an Fc protein comprising analkyne-containing non-naturally encoded amino acid is modified with aPEG derivative that contains a terminal azide or terminal alkyne moietythat is linked to the PEG backbone by means of an amide linkage.

In some embodiments, the alkyne-terminal PEG derivative will have thefollowing structure: RO—(CH₂CH₂O)_(n)—O—(CH₂)_(m)—NH—C(O)—(CH₂)_(p)—C═CHwhere R is a simple alkyl (methyl, ethyl, propyl, etc.), m is 2-10, p is2-10 and n is 100-1,000.

In another embodiment of the invention, an antigen-binding polypeptidecomprising an azide-containing amino acid is modified with a branchedPEG derivative that contains a terminal alkyne moiety, with each chainof the branched PEG having a MW ranging from 10-40 kDa and, morepreferably, from 5-20 kDa. For instance, in some embodiments, thealkyne-terminal PEG derivative will have the following structure:[RO—(CH₂CH₂O)_(n)—O—(CH₂)₂—NH—C(O)]₂CH(CH—₂)_(m)—X—(CH₂)_(p)C═CH where Ris a simple alkyl (methyl, ethyl, propyl, etc.), m is 2-10, p is 2-10,and n is 100-1,000, and X is optionally an O, N, S or carbonyl group(C═O), or not present.

In another embodiment of the invention, an Fc protein is modified with aPEG derivative that contains an activated functional group (includingbut not limited to, ester, carbonate) further comprising an arylphosphine group that will react with an azide moiety present on the sidechain of the non-naturally encoded amino acid. In general, the PEGderivatives will have an average molecular weight ranging from 1-100 kDaand, in some embodiments, from 10-40 kDa.

Other exemplary PEG molecules that may be linked to Fc proteins, as wellas PEGylation methods include those described in, e.g., U.S. PatentPublication Nos. 2004/0001838; 2002/0052009; 2003/0162949; 2004/0013637;2003/0228274; 2003/0220447; 2003/0158333; 2003/0143596; 2003/0114647;2003/0105275; 2003/0105224; 2003/0023023; 2002/0156047; 2002/0099133;2002/0086939; 2002/0082345; 2002/0072573; 2002/0052430; 2002/0040076;2002/0037949; 2002/0002250; 2001/0056171; 2001/0044526; 2001/0027217;2001/0021763; U.S. Pat. Nos. 6,646,110; 5,824,778; 5,476,653; 5,219,564;5,629,384; 5,736,625; 4,902,502; 5,281,698; 5,122,614; 5,473,034;5,516,673; 5,382,657; 6,552,167; 6,610,281; 6,515,100; 6,461,603;6,436,386; 6,214,966; 5,990,237; 5,900,461; 5,739,208; 5,672,662;5,446,090; 5,808,096; 5,612,460; 5,324,844; 5,252,714; 6,420,339;6,201,072; 6,451,346; 6,306,821; 5,559,213; 5,612,460; 5,747,646;5,834,594; 5,849,860; 5,980,948; 6,004,573; 6,129,912; WO 97/32607, EP229,108, EP 402,378, WO 92/16555, WO 94/04193, WO 94/14758, WO 94/17039,WO 94/18247, WO 94/28024, WO 95/00162, WO 95/11924, WO 95/13090, WO95/33490, WO 96/00080, WO 97/18832, WO 98/41562, WO 98/48837, WO99/32134, WO 99/32139, WO 99/32140, WO 96/40791, WO 98/32466, WO95/06058, EP 439 508, WO 97/03106, WO 96/21469, WO 95/13312, EP 921 131,WO 98/05363, EP 809 996, WO 96/41813, WO 96/07670, EP 605 963, EP 510356, EP 400 472, EP 183 503 and EP 154 316, which are incorporated byreference herein. Any of the PEG molecules described herein may be usedin any form, including but not limited to, single chain, branched chain,multiarm chain, single functional, bi-functional, multi-functional, orany combination thereof.

In certain embodiments, the Fc proteins can be linked to the payloadswith one or more linkers capable of reacting with the non-natural aminoacid. The one or more linkers can be any linkers apparent to those ofskill in the art. The term “linker” is used herein to refer to groups orbonds that normally are formed as the result of a chemical reaction andtypically are covalent linkages. Hydrolytically stable linkages meansthat the linkages are substantially stable in water and do not reactwith water at useful pH values, including but not limited to, underphysiological conditions for an extended period of time, perhaps evenindefinitely. Hydrolytically unstable or degradable linkages mean thatthe linkages are degradable in water or in aqueous solutions, includingfor example, blood. Enzymatically unstable or degradable linkages meanthat the linkage can be degraded by one or more enzymes. As understoodin the art, PEG and related polymers may include degradable linkages inthe polymer backbone or in the linker group between the polymer backboneand one or more of the terminal functional groups of the polymermolecule. For example, ester linkages formed by the reaction of PEGcarboxylic acids or activated PEG carboxylic acids with alcohol groupson a biologically active agent generally hydrolyze under physiologicalconditions to release the agent. Other hydrolytically degradablelinkages include, but are not limited to, carbonate linkages; iminelinkages resulted from reaction of an amine and an aldehyde; phosphateester linkages formed by reacting an alcohol with a phosphate group;hydrazone linkages which are reaction product of a hydrazide and analdehyde; acetal linkages that are the reaction product of an aldehydeand an alcohol; orthoester linkages that are the reaction product of aformate and an alcohol; peptide linkages formed by an amine group,including but not limited to, at an end of a polymer such as PEG, and acarboxyl group of a peptide; and oligonucleotide linkages formed by aphosphoramidite group, including but not limited to, at the end of apolymer, and a 5′ hydroxyl group of an oligonucleotide. Branched linkersmay be used in Fc proteins of the invention. A number of differentcleavable linkers are known to those of skill in the art. See U.S. Pat.Nos. 4,618,492; 4,542,225, and 4,625,014. The mechanisms for release ofan agent from these linker groups include, for example, irradiation of aphotolabile bond and acid-catalyzed hydrolysis. U.S. Pat. No. 4,671,958,for example, includes a description of immunoconjugates comprisinglinkers which are cleaved at the target site in vivo by the proteolyticenzymes of the patient's complement system. The length of the linker maybe predetermined or selected depending upon a desired spatialrelationship between the Fc protein and the molecule linked to it. Inview of the large number of methods that have been reported forattaching a variety of radiodiagnostic compounds, radiotherapeuticcompounds, drugs, toxins, and other agents to Fc proteins one skilled inthe art will be able to determine a suitable method for attaching agiven agent to an Fc protein.

Any hetero- or homo-bifunctional linker can be used to link theconjugates. The linker may have a wide range of molecular weight ormolecular length. Larger or smaller molecular weight linkers may be usedto provide a desired spatial relationship or conformation between the Fcprotein and the linked entity. Linkers having longer or shortermolecular length may also be used to provide a desired space orflexibility between the Fc protein and the linked entity. Similarly, alinker having a particular shape or conformation may be utilized toimpart a particular shape or conformation to the Fc protein or thelinked entity, either before or after the Fc protein reaches its target.The functional groups present on each end of the linker may be selectedto modulate the release of an Fc protein or a payload under desiredconditions. This optimization of the spatial relationship between the Fcprotein and the linked entity may provide new, modulated, or desiredproperties to the molecule.

In some embodiments, the invention provides water-soluble bifunctionallinkers that have a dumbbell structure that includes: a) an azide, analkyne, a hydrazine, a hydrazide, a hydroxylamine, or acarbonyl-containing moiety on at least a first end of a polymerbackbone; and b) at least a second functional group on a second end ofthe polymer backbone. The second functional group can be the same ordifferent as the first functional group. The second functional group, insome embodiments, is not reactive with the first functional group. Theinvention provides, in some embodiments, water-soluble compounds thatcomprise at least one arm of a branched molecular structure. Forexample, the branched molecular structure can be dendritic.

Fc Protein Compositions

Fc proteins and conjugates described herein can be formulated intocompositions using methods available in the art and those disclosedherein. Any of the compounds disclosed herein can be provided in theappropriate pharmaceutical composition and be administered by a suitableroute of administration.

In certain embodiments, the Fc protein or Fc conjugate compositionsprovided herein further comprise a pharmaceutically acceptable carrier.The carrier can be a diluent, excipient, or vehicle with which thepharmaceutical composition is administered. Such pharmaceutical carrierscan be sterile liquids, such as water and oils, including those ofpetroleum, animal, vegetable or synthetic origin, such as peanut oil,soybean oil, mineral oil, sesame oil and the like. Saline solutions andaqueous dextrose and glycerol solutions can also be employed as liquidcarriers, particularly for injectable solutions. Suitable pharmaceuticalexcipients include starch, glucose, lactose, sucrose, gelatin, malt,rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate,talc, sodium chloride, dried skim milk, glycerol, propylene, glycol,water, ethanol and the like. The composition, if desired, can alsocontain minor amounts of wetting or emulsifying agents, or pH bufferingagents. These compositions can take the form of solutions, suspensions,emulsion, tablets, pills, capsules, powders, sustained-releaseformulations and the like. Oral formulation can include standardcarriers such as pharmaceutical grades of mannitol, lactose, starch,magnesium stearate, sodium saccharine, cellulose, magnesium carbonate,etc. Examples of suitable pharmaceutical carriers are described in E.W.Martin, 1990, Remington's Pharmaceutical Sciences, Mack Publishing Co.

In some embodiments, the pharmaceutical composition is provided in aform suitable for administration to a human subject. In someembodiments, the pharmaceutical composition will contain aprophylactically or therapeutically effective amount of the Fc proteintogether with a suitable amount of carrier so as to provide the form forproper administration to the patient. The formulation should suit themode of administration.

In some embodiments, the pharmaceutical composition is provided in aform suitable for intravenous administration. Typically, compositionssuitable for intravenous administration are solutions in sterileisotonic aqueous buffer. Where necessary, the composition may alsoinclude a solubilizing agent and a local anesthetic such as lignocaineto ease pain at the site of the injection. Such compositions, however,may be administered by a route other than intravenous administration.

In particular embodiments, the pharmaceutical composition is suitablefor subcutaneous administration. In particular embodiments, thepharmaceutical composition is suitable for intramuscular administration.

Components of the pharmaceutical composition can be supplied eitherseparately or mixed together in unit dosage form, for example, as a drylyophilized powder or water free concentrate. Where the composition isto be administered by infusion, it can be dispensed with an infusionbottle containing sterile pharmaceutical grade water or saline. Wherethe composition is administered by injection, an ample of sterile waterfor injection or saline can be provided so that the ingredients may bemixed prior to administration.

In some embodiments, the pharmaceutical composition is supplied as a drysterilized lyophilized powder that is capable of being reconstituted tothe appropriate concentration for administration to a subject. In someembodiments, Fc proteins are supplied as a water free concentrate. Insome embodiments, the Fc protein is supplied as a dry sterilelyophilized powder at a unit dosage of at least 0.5 mg, at least 1 mg,at least 2 mg, at least 3 mg, at least 5 mg, at least 10 mg, at least 15mg, at least 25 mg, at least 30 mg, at least 35 mg, at least 45 mg, atleast 50 mg, at least 60 mg, or at least 75 mg.

In another embodiment, the pharmaceutical composition is supplied inliquid form. In some embodiments, the pharmaceutical composition isprovided in liquid form and is substantially free of surfactants and/orinorganic salts. In some embodiments, the Fc protein is supplied as inliquid form at a unit dosage of at least 0.1 mg/ml, at least 0.5 mg/ml,at least 1 mg/ml, at least 2.5 mg/ml, at least 3 mg/ml, at least 5mg/ml, at least 8 mg/ml, at least 10 mg/ml, at least 15 mg/ml, at least25 mg/ml, at least 30 mg/ml, or at least 60 mg/ml.

In some embodiments, the pharmaceutical composition is formulated as asalt form. Pharmaceutically acceptable salts include those formed withanions such as those derived from hydrochloric, phosphoric, acetic,oxalic, tartaric acids, etc., and those formed with cations such asthose derived from sodium, potassium, ammonium, calcium, ferrichydroxides, isopropylamine, triethylamine, 2-ethylamino ethanol,histidine, procaine, etc.

In therapeutic use, the practitioner will determine the posology mostappropriate according to a preventive or curative treatment andaccording to the age, weight, stage of the infection and other factorsspecific to the subject to be treated. In certain embodiments, doses arefrom about 1 to about 1000 mg per day for an adult, or from about 5 toabout 250 mg per day or from about 10 to 50 mg per day for an adult. Incertain embodiments, doses are from about 5 to about 400 mg per day or25 to 200 mg per day per adult. In certain embodiments, dose rates offrom about 50 to about 500 mg per day are also contemplated.

Methods of Use for Therapy or Prophylaxis

Certain Fc proteins or Fc protein conjugates provided herein can be usedfor the treatment or prevention of any disease or condition deemedsuitable to the practitioner of skill in the art. Generally, a method oftreatment or prevention encompasses the administration of atherapeutically or prophylactically effective amount of the Fc proteinor conjugate composition to a subject in need thereof to treat orprevent the disease or condition.

A therapeutically effective amount of the Fc protein or conjugatecomposition is an amount that is effective to reduce the severity, theduration and/or the symptoms of a particular disease or condition. Theamount of the Fc protein or composition that will be therapeuticallyeffective in the prevention, management, treatment and/or ameliorationof a particular disease can be determined by standard clinicaltechniques. The precise amount of the Fc protein or composition to beadministered with depend, in part, on the route of administration, theseriousness of the particular disease or condition, and should bedecided according to the judgment of the practitioner and each subject'scircumstances.

In some embodiments, the effective amount of the Fc protein or conjugateprovided herein is between about 0.025 mg/kg and about 1000 mg/kg bodyweight of a human subject. In certain embodiments, the Fc protein isadministered to a human subject at an amount of about 1000 mg/kg bodyweight or less, about 950 mg/kg body weight or less, about 900 mg/kgbody weight or less, about 850 mg/kg body weight or less, about 800mg/kg body weight or less, about 750 mg/kg body weight or less, about700 mg/kg body weight or less, about 650 mg/kg body weight or less,about 600 mg/kg body weight or less, about 550 mg/kg body weight orless, about 500 mg/kg body weight or less, about 450 mg/kg body weightor less, about 400 mg/kg body weight or less, about 350 mg/kg bodyweight or less, about 300 mg/kg body weight or less, about 250 mg/kgbody weight or less, about 200 mg/kg body weight or less, about 150mg/kg body weight or less, about 100 mg/kg body weight or less, about 95mg/kg body weight or less, about 90 mg/kg body weight or less, about 85mg/kg body weight or less, about 80 mg/kg body weight or less, about 75mg/kg body weight or less, about 70 mg/kg body weight or less, or aboutmg/kg body weight or less.

In some embodiments, the effective amount of Fc protein or conjugateprovided herein is between about 0.025 mg/kg and about 60 mg/kg bodyweight of a human subject. In some embodiments, the effective amount ofan Fc protein of the pharmaceutical composition provided herein is about0.025 mg/kg or less, about 0.05 mg/kg or less, about 0.10 mg/kg or less,about 0.20 mg/kg or less, about 0.40 mg/kg or less, about 0.80 mg/kg orless, about 1.0 mg/kg or less, about 1.5 mg/kg or less, about 3 mg/kg orless, about 5 mg/kg or less, about 10 mg/kg or less, about 15 mg/kg orless, about 20 mg/kg or less, about 25 mg/kg or less, about 30 mg/kg orless, about 35 mg/kg or less, about 40 mg/kg or less, about 45 mg/kg orless, about 50 mg/kg or about 60 mg/kg or less.

The pharmaceutical composition of the method can be administered usingany method known to those skilled in the art. For example, thepharmaceutical composition can be administered intramuscularly,intradermally, intraperitoneally, intravenously, subcutaneouslyadministration, or any combination thereof. In some embodiments, thepharmaceutical composition is administered subcutaneously. In someembodiments, the composition is administered intravenously. In someembodiments, the composition is administered intramuscularly.

Methods of Use for Detection or Diagnosis

The Fc proteins of Fc protein conjugates provided herein can be used forthe detection of any target or for the diagnosis of any disease orcondition deemed suitable to the practitioner of skill in the art. Themethods encompass detecting the binding of an Fc protein to a targetantigen in the appropriate location, e.g., the appropriate body, tissue,or cell. In the methods, the formation of a complex between the Fcprotein and antigen can be detected by any method known to those ofskill in the art. Examples include assays that use secondary reagentsfor detection, ELISA's and immunoprecipitation and agglutination assays.A detailed description of these assays is, for example, given in Harlowand Lane, Antibodies: A Laboratory Manual (Cold Spring HarborLaboratory, New York 1988 555-612, WO96/13590 to Maertens and Stuyver,Zrein et al. (1998) and WO96/29605.

For in situ diagnosis, the Fc protein or conjugate may be administeredto a subject by methods known in the art such as, for example,intravenous, intranasal, intraperitoneal, intracerebral, intraarterialinjection such that a specific binding between an Fc protein accordingto the invention with an eptitopic region on the amyloid protein mayoccur. The Fc protein/antigen complex may conveniently be detectedthrough a label attached to the Fc protein or any other art-known methodof detection.

Further provided herein are kits for detection or diagnosis. Exemplarykits comprise one or more Fc proteins or conjugates provided hereinalong with one or more reagents useful for detecting a complex betweenthe one or more Fc proteins and their target antigens.

Preparation of Fc proteins

The Fc proteins described herein can be prepared by any techniqueapparent to those of skill in the art without limitation. Usefultechniques for preparation include in vivo synthesis, for example withmodified tRNA and tRNA synthetase, cell-free synthesis, for example withmodified tRNA and tRNA synthetase, solid phase polypeptide synthesis andliquid phase polypeptide synthesis. Exemplary techniques are describedin this section and in the examples below.

In certain methods, the Fc protein is translated and/or transcribed fromone or more polynucleotides encoding the polypeptide chains of the Fcprotein. Accordingly, provided herein are polynucleotides capable ofencoding the Fc proteins having one or more non-natural amino acids atsite-specific positions in one or more polypeptide chains. In certainembodiments, the polynucleotides comprise a codon not normallyassociated with an amino acid at the polynucleotide positioncorresponding to the site-specific polypeptide position for thenon-natural amino acid. Examples of such codons include stop codons, 4bp codons, 5 bp codons, and the like. The reaction mixture typicallycomprises a tRNA synthetase capable of making tRNAs that complement(suppress) corresponding codons. These suppressor tRNAs are linked tothe non-natural amino acids to facilitate their incorporation into thepolypeptide at the site of the suppressor codon.

The Fc proteins can be prepared by techniques known to those of skill inthe art for expressing such polynucleotides to incorporate non-naturalamino acids into site specific positions of a polypeptide chain. Suchtechniques are described, for example, in U.S. Pat. Nos. 7,045,337 and7,083,970, in U.S. Published Patent Application Nos. US 2008/0317670, US2009/0093405, US 2010/0093082, US 2010/0098630, US 2008/0085277 and ininternational patent publication nos. WO 2004/016778 A1 and WO2008/066583 A2, the contents of which are hereby incorporated byreference in their entireties.

In certain embodiments, an Fc protein can be prepared in a cell-freereaction mixture comprising at least one orthogonal tRNA aminoacylatedwith an unnatural amino acid, where the orthogonal tRNA base pairs witha codon that is not normally associated with an amino acid, e.g. a stopcodon; a 4 bp codon, etc. The reaction mixture also comprises a tRNAsynthetase capable of aminoacylating the orthogonal tRNA with anunnatural amino acid. Usually the orthogonal tRNA synthetase, which issusceptible to degradation by proteases present in bacterial cellextracts, is exogenously synthesized and added to the reaction mix priorto initiation of polypeptide synthesis. The orthogonal tRNA may besynthesized in the bacterial cells from which the cell extract isobtained, may be synthesized de novo during the polypeptide synthesisreaction, or may be exogenously added to the reaction mix.

In certain embodiments, components that affect unnatural amino acidinsertion and protein insertion or folding are optionally added to thereaction mixture. Such components include elevated concentrations oftranslation factors to minimize the effect of release factor 1 and 2 andto further optimize orthogonal component concentrations. Proteinchaperones (Dsb System of oxidoreductases and isomerases, GroES, GroEL,DNAJ, DNAK, Skp, etc.) may be exogenously added to the reaction mixtureor may be overexpressed in the source cells used to prepare the cellextract The reactions may utilize a large scale reactor, small scale, ormay be multiplexed to perform a plurality of simultaneous syntheses.Continuous reactions will use a feed mechanism to introduce a flow ofreagents, and may isolate the end-product as part of the process. Batchsystems are also of interest, where additional reagents may beintroduced to prolong the period of time for active synthesis. A reactormay be run in any mode such as batch, extended batch, semi-batch,semi-continuous, fed-batch and continuous, and which will be selected inaccordance with the application purpose. The reactions may be of anyvolume, either in a small scale, usually at least about 1 μl and notmore than about 15 μl, or in a scaled up reaction, where the reactionvolume is at least about 15 μl, usually at least about 50 μl, moreusually at least about 100 μl, and may be 500 μl, 1000 μl, or greater.In principle, reactions may be conducted at any scale as long assufficient oxygen (or other electron acceptor) is supplied when needed.

Useful methods for synthesis where at least one unnatural amino acid isintroduced into the polypeptide strand during elongation include but arenot limited to: (I) addition of exogenous purified orthogonalsynthetase, unnatural amino acid, and orthogonal tRNA to the cell-freereaction, (II) addition of exogenous purified orthogonal synthetase andunnatural amino acid to the reaction mixture, but with orthogonal tRNAtranscribed during the cell-free reaction, (III) addition of exogenouspurified orthogonal synthetase and unnatural amino acid to the reactionmixture, but with orthogonal tRNA synthesized by the cell extract sourceorganism. In certain embodiments, the orthogonal components are drivenby regulatable promoters, so that synthesis levels can be controlledalthough other measures may be used such as controlling the level of therelevant DNA templates by addition or specific digestion.

In some embodiments, a bacterial cell-free expression system is used toproduce protein or peptide variants with non-native amino acids (nnAA).The use of bacterial cell-free extracts for in vitro protein synthesisoffers several advantages over conventional in vivo protein expressionmethods. Cell-free systems can direct most, if not all, of the metabolicresources of the cell towards the exclusive production of one protein.Moreover, the lack of a cell wall and membrane components in vitro isadvantageous since it allows for control of the synthesis environment.However, the efficiency of cell-free extracts can be decreased bybacterial proteins that inhibit protein synthesis, either directly orindirectly. Thus, inactivation of undesirable proteins that decrease theefficiency of protein synthesis should increase the yield of desirableproteins in cell-free extracts. For example, the inactivation ofproteins that decrease the efficiency of protein synthesis shouldincrease the yield of polypeptides having non-native amino acidsincorporated at a defined amino acid residue. The introduction of nnAAinto polypeptides is useful for increasing the biological diversity andfunction of proteins. One approach for producing polypeptides having annAA incorporated at a defined amino acid residue is to use an nnAA,aminoacylated orthogonal CUA containing tRNA for introduction of thennAA into the nascent polypeptide at an amber (stop) codon duringprotein translation. However, the incorporation of nnAA at an ambercodon can be inhibited by the native bacterial termination complex,which normally recognizes the stop codon and terminates translation.Release Factor 1 (RF1) is a termination complex protein that facilitatesthe termination of translation by recognizing the amber codon in an mRNAsequence. RF1 recognition of the amber stop codon can promote pre-maturetruncation products at the site of non-native amino acid incorporation,and thus decreased protein yield. Therefore, attenuating the activity ofRF1 may increase nnAA incorporation into recombinant proteins.

It has previously been shown that nnAA incorporation can be increased byattenuating RF1 activity in 3 ways: 1) neutralizing antibodyinactivation of RF1, 2) genomic knockout of RF1 (in an RF2 bolsteredstrain), and 3) site specific removal of RF1 using a strain engineeredto express RF1 containing a protein tag for removal by affinitychromatography (Chitin Binding Domain and His Tag). Another method forinactivating RF1 comprises introducing proteolytic cleavage sites intothe RF1 amino acid sequence. The cleavage sites are not accessible tothe protease during bacterial cell growth, but are cleaved by theprotease when the bacterial cells are lysed to produce cell-freeextract. Thus, the yield of full length polypeptides having a nnAAincorporated at an amber codon is increased in bacterial cell extractsexpressing such modified RF1 variants.

In some embodiments, in order to produce Fc proteins comprising anon-natural amino acid, one needs a nucleic acid template. The templatesfor cell-free protein synthesis can be either mRNA or DNA. The templatecan comprise sequences for any particular antibody of interest, and mayencode a full-length antibody or a fragment of any length thereof.Nucleic acids that serve as protein synthesis templates are optionallyderived from a natural source or they can be synthetic or recombinant.For example, DNAs can be recombinant DNAs, e.g., plasmids, viruses orthe like.

In some embodiments, once a nucleic acid template of an Fc protein isproduced, the template is used to synthesize the antibody in a cell-freetranslation system. For example, the template can be added to a celllysate under conditions sufficient to translate the template intoprotein. The cell lysate can be from bacterial cells or eukaryoticcells. The expressed Fc protein can then be purified using methods knownin the art, as described below.

In some embodiments, a translation system (e.g., an in vitro proteinsynthesis system) is used to produce the Fc protein with one or morennAAs incorporated therein. An exemplary translation system comprises acell free extract, cell lysate, or reconstituted translation system,along with the nucleic acid template for synthesis of the desiredpolypeptide or protein having non-native amino acids at preselected(defined) positions. The reaction mixture will further comprise monomersfor the macromolecule to be synthesized, e.g., amino acids, nucleotides,etc., and such co-factors, enzymes and other reagents that are necessaryfor the synthesis, e.g., ribosomes, tRNA, polymerases, transcriptionalfactors, etc. In addition to the above components such as a cell-freeextract, nucleic acid template, and amino acids, materials specificallyrequired for protein synthesis may be added to the reaction. Thematerials include salts, folinic acid, cyclic AMP, inhibitors forprotein or nucleic acid degrading enzymes, inhibitors or regulators ofprotein synthesis, adjusters of oxidation/reduction potentials,non-denaturing surfactants, buffer components, spermine, spermidine,putrescine, etc. Various cell-free synthesis reaction systems are wellknown in the art. See, e.g., Kim, D. M. and Swartz, J. R. Biotechnol.Bioeng. 66:180-8 (1999); Kim, D. M. and Swartz, J. R. Biotechnol. Prog.16:385-90 (2000); Kim, D. M. and Swartz, J. R. Biotechnol. Bioeng.74:309-16 (2001); Swartz et al, Methods Mol. Biol. 267:169-82 (2004);Kim, D. M. and Swartz, J. R. Biotechnol. Bioeng. 85:122-29 (2004);Jewett, M. C. and Swartz, J. R., Biotechnol. Bioeng. 86:19-26 (2004);Yin, G. and Swartz, J. R., Biotechnol. Bioeng. 86:188-95 (2004); Jewett,M. C. and Swartz, J. R., Biotechnol. Bioeng. 87:465-72 (2004); Voloshin,A. M. and Swartz, J. R., Biotechnol. Bioeng. 91:516-21 (2005).Additional conditions for the cell-free synthesis of desiredpolypeptides are described in WO2010/081 110, the contents of which areincorporated by reference herein in its entirety.

In some embodiments, a DNA template is used to drive in vitro proteinsynthesis, and RNA polymerase is added to the reaction mixture toprovide enhanced transcription of the DNA template. RNA polymerasessuitable for use herein include any RNA polymerase that functions in thebacteria from which the bacterial extract is derived. In otherembodiments, an RNA template is used to drive in vitro proteinsynthesis, and the components of the reaction mixture can be admixedtogether in any convenient order, but are preferably admixed in an orderwherein the RNA template is added last, thereby minimizing potentialdegradation of the RNA template by nucleases.

In some embodiments, a cell-free translation system is used to producethe antibody with one or more nnAAs incorporated therein. Cell-freeprotein synthesis exploits the catalytic power of the cellularmachinery. Obtaining maximum protein yields in vitro requires adequatesubstrate supply, e.g., nucleoside triphosphates and amino acids, ahomeostatic environment, catalyst stability, and the removal oravoidance of inhibitory byproducts. The optimization of in vitrosynthetic reactions benefits from recreating the in vivo state of arapidly growing organism. In some embodiments of the invention,cell-free synthesis is therefore performed in a reaction where oxidativephosphorylation is activated. Additional details are described in U.S.Pat. No. 7,338,789, the contents of which are incorporated by referenceherein in its entirety.

In vitro, or cell-free, protein synthesis offers several advantages overconventional in vivo protein expression methods. Cell-free systems candirect most, if not all, of the metabolic resources of the cell towardsthe exclusive production of one protein. Moreover, the lack of a cellwall and membrane components in vitro is advantageous since it allowsfor control of the synthesis environment. For example, tRNA levels canbe changed to reflect the codon usage of genes being expressed. Theredox potential, pH, or ionic strength can also be altered with greaterflexibility than with in vivo protein synthesis because concerns of cellgrowth or viability do not exist. Furthermore, direct recovery ofpurified, properly folded protein products can be easily achieved. Insome embodiments, the productivity of cell-free systems has improvedover 2-orders of magnitude in recent years, from about 5 μg/ml-hr toabout 500 μg/ml-hr.

In certain embodiments, tRNA synthetase is exogenously synthesized andadded to the cell-free reaction mix. In certain embodiments, thereaction mix is prepared from bacterial cells in which ompT has beeninactivated or is naturally inactive. OmpT is believed to degradecomponents of the reaction mixture including tRNA synthetase.

In addition to the above components such as cell-free extract, genetictemplate, and amino acids, materials specifically required for proteinsynthesis may be added to the reaction. These materials include salts,folinic acid, cyclic AMP, inhibitors for protein or nucleic aciddegrading enzymes, inhibitors or regulators of protein synthesis,adjusters of oxidation/reduction potential(s), non-denaturingsurfactants, buffer components, spermine, spermidine, putrescine, etc.

The salts preferably include potassium, magnesium, and ammonium salts(e.g. of acetic acid or glutamic acid). One or more of such salts mayhave an alternative amino acid as a counter anion. There is aninterdependence among ionic species for optimal concentration. Theseionic species are typically optimized with regard to protein production.When changing the concentration of a particular component of thereaction medium, that of another component may be changed accordingly.For example, the concentrations of several components such asnucleotides and energy source compounds may be simultaneously adjustedin accordance with the change in those of other components. Also, theconcentration levels of components in the reactor may be varied overtime. The adjuster of oxidation/reduction potential may bedithiothreitol, ascorbic acid, glutathione and/or their oxidized forms.

In certain embodiments, the reaction can proceed in a dialysis mode, ina diafiltration batch mode, in a fed-batch mode of in a semi-continuousoperation mode. In certain embodiments, a feed solution can be suppliedto the reactor through a membrane or through an injection unit.Synthesized Fc protein can accumulate in the reactor followed byisolation or purification after completion of the system operation.Vesicles containing the Fc protein may also be continuously isolated,for example by affinity adsorption from the reaction mixture either insitu or in a circulation loop as the reaction fluid is pumped past theadsorption matrix.

During protein synthesis in the reactor, the protein isolating means forselectively isolating the desired protein may include a unit packed withparticles coated with Fc protein molecules or other molecules foradsorbing the synthesized, desired protein. Preferably, the proteinisolating means comprises two columns for alternating use.

The resulting Fc protein can be purified or isolated by standardtechniques. Exemplary techniques are provided in the examples below.

Assay Methods

Fc proteins can be assayed for their expected activity, or for a newactivity, according to any assay apparent to those of skill in the art.The resulting Fc protein can be assayed activity in a functional assay,e.g. binding to Fc receptor, or by quantitating the amount of proteinpresent in a non-functional assay, e.g. immunostaining, ELISA,quantitation on Coomasie or silver stained gel, etc., and determiningthe ratio of biologically active protein to total protein.

The amount of protein produced in a translation reaction can be measuredin various fashions. One method relies on the availability of an assaywhich measures the activity of the particular protein being translated.An example of an assay for measuring protein activity is a luciferaseassay system, or chloramphenical acetyl transferase assay system. Theseassays measure the amount of functionally active protein produced fromthe translation reaction. Activity assays will not measure full lengthprotein that is inactive due to improper protein folding or lack ofother post translational modifications necessary for protein activity.

Another method of measuring the amount of protein produced in coupled invitro transcription and translation reactions is to perform thereactions using a known quantity of radiolabeled amino acid such as³⁵S-methionine, ³H-leucine or ¹⁴C-leucine and subsequently measuring theamount of radiolabeled amino acid incorporated into the newly translatedprotein. Incorporation assays will measure the amount of radiolabeledamino acids in all proteins produced in an in vitro translation reactionincluding truncated protein products. The radiolabeled protein may befurther separated on a protein gel, and by autoradiography confirmedthat the product is the proper size and that secondary protein productshave not been produced.

EXAMPLES

As used herein, the symbols and conventions used in these processes,schemes and examples, regardless of whether a particular abbreviation isspecifically defined, are consistent with those used in the contemporaryscientific literature, for example, the Journal of Biological Chemistry.

For all of the following examples, standard work-up and purificationmethods known to those skilled in the art can be utilized. Unlessotherwise indicated, all temperatures are expressed in ° C. (degreesCentigrade). All methods are conducted at room temperature unlessotherwise noted. The follow examples set exemplary Fc-containingmolecules containing non-natural amino acids that are in the context offull-length antibody sequences. It is believed that the N-terminal heavychain sequences and light chain sequences do not significantly affectthe suitability of the particular sites for incorporation of anon-natural amino acid and/or conjugation to that non-natural aminoacid. Thus, sites that have desirable properties in the context of afull-length antibody also have desirable properties in the context of anFc protein.

Example 1 Synthesis of Exemplary Antibodies Containing Non-Natural AminoAcids and Conjugation to an Exemplary Cytotoxic Agent

The following example describes exemplary antibodies containingnon-natural amino acids at defined positions along with methods of theirconstruction and expression. The antibodies were assessed forexpression, suppression efficiency, efficiency of conjugation with acell-killing agent, cell binding, and cell killing, as set forth below.In this example, the exemplary antibodies are based on parent antibodytrastuzumab (U.S. Pat. Nos. 6,165,464 and 2006/0018899 A1; Carter etal., 1992, Proc. Natl. Acad. Sci. USA 10:4285-4289).

First, constructs were made incorporating a TAG amber codon at a definedposition in the heavy chain of trastuzumab. Site directed mutagenesiswas performed using a pYD plasmid containing the coding region oftrastuzumab with a C-terminal-(His)₆ tag (SD02005) as DNA template andsynthetic oligonucleotides (Eurofins MWS Operon; Huntsville, Ala.)containing mutations of interest in both sense and antisense directions.Oligonucleotides of each mutation were added to the DNA template andPHUSION® polymerase (New England Biolabs; Ipswich, Mass.) to a finalvolume of 20 μL. The final concentration of each component was 0.16 μMof each oligonucleotide, 0.5 ng/μL template DNA, 0.02 U/μL PHUSION®polymerase in HF buffer containing 1.5 mM MgCl₂ and 200 μM dNTP. Mixturewas incubated at 98° C. 5 m, 18 PCR cycles (98° C. 30 s, 55° C. 1 min,72° C. 4 min), 10 min at 72° C. and stored at 4° C. DpnI (New EnglandBiolabs; Ipswich, Mass.) was added to the mixture to final concentrationof 0.6 U/μL and incubated for 37° C. 1 h to digest parent DNA.

5 μL of each mixture was then transformed into 50 μL of chemicallycompetent E. coli cells with a MultiShot™ 96-Well Plate TOP10 accordingto the manufacturer's procedure (Invitrogen; Carlsbad, Calif.).Transformed cells were recovered in 200 μL SOC (Invitrogen; Carlsbad,Calif.) at 37° C. for 1 hr and plated onto Luria-Bertani (LB) agarsupplemented with 50 μg/mL kanamycin (Teknova; Hollister, Calif.). After24 hrs at 37° C., colonies were picked into 200 μL LB with 7.5% glyceroland 50 μg/mL kanamycin, and grown at 37° C. for 24 hrs. 20 μL of culturewas used for rolling circle amplification (RCA) and sequenced by primerextension using T7 (SEQ ID NO: 2, 5′TAATACGACTCACTATAGG 3′) and T7 term(SEQ ID NO: 3, 5′GCTAGTTATTGCTCAGCG3′) primers. Sequence was analyzedusing SEQUENCHER® software (Gene Codes Corp; Ann Arbor, Mich.), andclones containing mutations were picked and arrayed into 96 well plates.Overnight cultures of selected variants were grown and used forpreparing plasmid DNA using the standard 96 well mini-prep protocolaccording to the manufacturer (Qiagen; Germantown, Md.). Concentrationof mini-prepped DNA was measured using absorbance at 260 nm (and 280nm).

The variants were then expressed in a cell-free protein synthesisreaction as follows as described in Zawada et al., 2011, Biotechnol.Bioeng. 108(7)1570-1578 with the modifications described below.Unsubstituted trastuzumab was also made as a control. Cell-free extractswere treated with 50 μM iodoacetamide for 30 min at RT (20° C.) andadded to a premix containing all other components except for heavy chainDNA from variants of interest. Cell free reactions were initiated byaddition of plasmid DNA of heavy chain DNA variants and incubated at 30°C. for 12 h on a shaker at 450 rpm in 96 deep well plates. The reactionwas incubated further at 4° C. for 5 h. The final concentration in theprotein synthesis reaction was 30% cell extract, 1 mM para-azidophenylalanine (pAzF) (RSP Amino Acids), 0.125 mg/mL M. jannaschii ambersuppressor tRNA, 0.37 mg/mL M. jannaschii pAzF-specific amino-acyl tRNAsynthetase (FRS), 2 mM GSSG, 0.29 mg/mL PDI (Mclab), 100 μg/mL E. coliDsbC, 8 mM magnesium glutamate, 10 mM ammonium glutamate, 130 mMpotassium glutamate, 35 mM sodium pyruvate, 1.2 mM AMP, 0.86 mM each ofGMP, UMP, and CMP, 2 mM amino acids (except 0.5 mM for Tyrosine andPhenylalanine), 4 mM sodium oxalate, 1 mM putrescine, 1.5 mM spermidine,15 mM potassium phosphate, 100 nM T7 RNAP, 2 μg/mL trastuzumab lightchain DNA, 8 μg/mL trastuzumab-(His)₆ heavy chain DNA. Each trastuzamabvariant was produced in 1 mL scale in 96 deep well plates in duplicates.A total of three plates were used to express the variants, each comparedto expression of an unsubstituted trastuzumab to normalize forexpression across the plates. It should be noted that all trastuzumabvariants thus produced were aglycosylated.

To monitor protein synthesis, a portion of each cell-free proteinsynthesis reaction was removed and spiked with 3.33% (v/v)l-[U-14C]-leucine (300 mCi/mmole; GE Life Sciences, Piscataway, N.J.).The suppression of amber codon at different sites of the heavy chain wasdetermined by [¹⁴C]-autoradiograhy of reducing SDS-PAGE gels. Fulllength trastuzumab heavy chain and suppressed tastuzumab heavy chainvariants run at 50 kD on SDS-PAGE. Non suppressed (truncated)trastuzumab variants run at a lower molecular weight. Amber suppressionin the heavy chain is determined by:

${suppression} = \frac{{band}\mspace{14mu}{intensity}\mspace{14mu}{of}\mspace{14mu}{suppressed}\mspace{14mu}{heavy}\mspace{14mu}{chain}\mspace{14mu}{TAG}\mspace{14mu}{variant}}{{band}\mspace{14mu}{intensity}\mspace{14mu}{of}\mspace{14mu}{wild}\mspace{14mu}{type}\mspace{14mu}{heavy}\mspace{14mu}{chain}}$

Band intensity was determined by ImageQuant (Amersham Biosciences Corp.;Piscataway, N.J.). It should be noted that this 14C gel assay cannot beused to assess suppressions for TAG variants of heavy chain from theposition of 425 (EU index numbering) to C terminus since the truncatedvariants cannot be distinguished from the full length product.

Following synthesis, each 1 mL reaction was diluted with 1 mL PBS atpH7.4. The mixture was centrifuged at 5000×g 4° C. for 15 min.Supernatant was captured with IMAC Phytip containing 40 μL resin(PhyNexus, Inc.; San Jose, Calif.) by pipetting up and down 4 timesslowly at a flow rate of 4.2 μl/min. Resin was washed with 925 μL IMACbinding buffer (50 mM Tris pH 8.0, 300 mM NaCl, 10 mM imidazole) bypipetting up and down twice at a flow rate of 8.3 μL/min. This processwas repeated once with an additional 925 μL IMAC binding buffer. Boundprotein was eluted with 250 μL IMAC elution buffer (50 mM Tris pH 8.0,300 mM NaCl, 500 mM Imidazole) by pipetting up and down 4 times at aflow rate of 4.2 μL/min. As the (his)6 tag of the construct isC-terminal, this procedure allows isolation of full length protein. Toquantitate the variants following purification, IMAC purified sampleswere mixed with 2× gel loading buffer (Bio-Rad#161-0737) resolved by of4-15% Stain-Free™ gel (Bio-Rad Criterion™ TGX #567-8085). Samples werenot heated before loading into wells of the gel. Protein bands werevisualized and quantitated by Bio-Rad Gel Doc EZ System using Image Labsoftware (v3). Band intensities of samples were determined based on massstandards of HERCEPTIN® loaded on the same gel.

Next, the trastuzumab variants were conjugated to an exemplary cytotoxicagent, MMAF, using a constrained cyclooctyne reagent. In brief,DBCO-MMAF (structure shown as FIG. 1; ACME Bioscience; Palo Alto,Calif.) was dissolved in DMSO to a final concentration of 5 mM. Thecompound was diluted into with PBS to 1 mM and then added totrastuzumab-(His)₆ variants in IMAC elution buffer to final drugconcentration of 100 μM. Mixture was incubated at RT (20° C.) for 17hours. Reaction was stopped by adding Sodium Azide to finalconcentration of 100 mM and buffer exchanged using Zeba plates (ThermoScientific; Waltham, Mass.) equilibrated in 1×PBS. Filtrate was thenpassed through a MUSTANG® Q plate (Pall Corp.; Port Washington, N.Y.) toremove endotoxin.

Example 2 Characterization of Exemplary Antibody-Drug Conjugates

Hydrophobic Interaction Chromatography (HIC) was performed to quantitatethe samples and to determine the drug-antibody ratios of the trastuzumabvariant drug conjugates as follows. Samples and standards were diluted1:2 in 3M Ammonium Sulfate (EMD Chemical), 50 mM Sodium Phosphate pH 7.0(Mallinckrodt) prepared in MilliQ water. An Agilent 1100 binary pumpHPLC system was equipped with a Tosoh Bioscience LLC TSK-gel Butyl-NPR®(4.6 mm×3.5 cm) column with a column compartment temperature of 30° C.The mobile phase A was 1.5M Ammonium Sulfate, 50 mM Sodium Phosphate, pH7.0. The mobile phase B was 50 mM Sodium Phosphate, pH 7.0 in 80:20water:Isopropyl Alcohol (Honeywell). The mobile phase was delivered at aflow rate of 1.0 mL/minute. The separation was performed with a lineargradient of 15% mobile phase B to 100% mobile phase B in 10 minutes. TheUV data was acquired at both 210 and 280 nm. The peak areas werequantitated using Chemstation software (Agilent) and the Drug AntibodyRatio (DAR) was calculated from the percent of the total peak area.

To assess binding of the conjugated trastuzumab variants, a bindingassay to cells expressing HER2 was performed as follows. The binding ofthe purified conjugated variants to HER2 on SKBR3 cells, whichoverexpress the HER2/c-erb-2 gene product, with over 1.5 millionreceptor copies per cell (ATCC #HTB-30, Manassas, Va.) was compared toclinical grade Herceptin®, unglycosylated trastuzumab produced bycell-free protein synthesis, or human serum IgG1 as a negative control(Sigma-Aldrich; St. Louis, Mo.). SKBR3 cells were cultured in DMEM:Ham'sF-12 (50:50), high glucose (Cellgro-Mediatech; Manassas, Va.)supplemented with 10% heat-inactivated fetal bovine serum (Hyclone;Thermo Scientific; Waltham, Mass.), 2 mM glutamax (Invitrogen; Carlsbad,Calif.) and 1× Pencillin/streptomycin (Cellgro-Mediatech; Manassas,Va.). Adherent cells were washed twice with calcium and magnesium-freeHanks Balanced Salt Solution (HBSS), harvested with HYQ®TASE™ (Hyclone;Thermo Scientific; Waltham, Mass.). A total of 200,000 cells per samplein total volume of 10 μL were incubated with serial dilutions of eitherconjugated trastuzumab variant, clinical grade HERCEPTIN®, oraglycosylated trastuzumab made in 10 μL FACS buffer (DPBS buffersupplemented with 1% bovine serum albumin). Cells plus antibody or ADCwere incubated for 60 minutes on ice. Unstained cells, human IgG1(Isotype control) and Secondary antibody (goat anti-human IgG) were usedas controls. Cells were washed twice with ice-cold FACS buffer andincubated with either 5 g/ml Alexa 647 labeled goat anti-human IgGsecondary antibody (Invitrogen; Carlsbad, Calif.) on ice for 1 hour. Allsamples were washed using FACS buffer and analyzed using a BD FACSCalibur system (BD Biosciences; San Jose, Calif.).

Mean fluorescence intensities were fitted using non-linear regressionanalysis with one site specific binding equation using GraphPad Prism(GraphPad v 5.00, Software; San Diego, Calif.). Data was expressed asRelative MFI vs. concentration of antibody or antibody variant in μg/ml.

Next, the effects of the conjugated trastuzumab on cell killing weremeasured with a cell proliferation assay as follows. SKBR3 andMDA-MB-468 were obtained from ATCC and maintained in DMEM:Ham's F-12(50:50), high glucose (Cellgro-Mediatech; Manassas, Va.) supplementedwith 10% heat-inactivated fetal bovine serum (Hyclone; ThermoScientific; Waltham, Mass.), 2 mM glutamax (Invitrogen; Carlsbad,Calif.) and 1× Pencillin/streptomycin (Cellgro-Mediatech; Manassas,Va.). Adherent cells were washed twice with calcium and magnesium-freeHanks Balanced Salt Solution (HBSS), harvested with HYQ®TASE™ (Hyclone;Thermo Scientific; Waltham, Mass.). A total of 10³ cells were seeded ina volume of 40 μl in a 96-well half area flat bottom white Polystyreneplate. The cells were allowed to adhere overnight at 37° C. in a CO₂incubator. ADC variants were formulated at 2× concentration in DMEM/F12medium and filtered through MultiScreen _(HTS) 96-Well Filter Plates(Millipore; Billerica, Mass.). Filter sterilized conjugated trastuzumabvariants, HERCEPTIN®, or aglycoslyated trastuzumab were added intotreatment wells and plates were cultured at 37° C. in a CO2 incubatorfor 120 hrs. For cell viability measurement, 80 μl of Cell Titer-Glo®reagent (Promega Corp.; Madison, Wis.) was added into each well, andplates processed as per product instructions. Relative luminescence wasmeasured on an ENVISION® plate reader (Perkin-Elmer; Waltham, Mass.).Relative luminescence readings were converted to % viability usinguntreated cells as controls. Data was fitted with non-linear regressionanalysis, using log(inhibitor) vs. response-Variable slope, 4-parameterfit equation using GraphPad Prism (GraphPad v 5.00, Software; San Diego,Calif.). Data was expressed as relative cell viability, ATP content %vs. dose of ADC in μg/ml.

Positive results of these characterization experiments are presented inTables 1, 2, and 3 (plates 1, 2, and 3, respectively). In the Tables,the following properties of the variants are shown: the rank order andconcentration of protein made, the efficiency of expression of fulllength product compared to wild-type (suppression efficiency), abilityto bind HER2-expressing cells, drug-antibody ratio (expressed as thenumber of cytotoxic agents per antibody), a comment regarding the HICprofile, and the observed IC₅₀ from the cell-killing assay describedabove. In the comments regarding the HIC assay, Single Peak correspondsto a profile that resolved well as a single peak (an example of such aprofile is shown as FIG. 2), NWR corresponds to a poorly resolvedprofile (an example of such a profile is shown as FIG. 3), and WRcorresponds to a well-resolved HIC profile (an example of such a profileis shown as FIG. 4). Several of the variants that exhibited a singlepeak profile also exhibited potent killing, which indicates that thevariant was indeed conjugated with drug, but failed to resolve on theHIC column.

In general, preferred variants exhibit an IC₅₀ of about 20 ng/ml orbelow, even more preferably below about 10 ng/ml, and even morepreferably below about 5 ng/ml. Preferably, the variants are in the top50% of expression, and even more preferably, are in the top 25% ofexpression. Variants that have a DAR of at least about 1.0 are alsopreferred. Preferred variants include those with a non-natural aminoacid replacing positions P238, S239, F241, F243, K246, E356, M358, K360,V262, V264, D265, S267, H268, E269, D270, P271, E272, K274, F275, Y278,D280, G281, V282, E283, N286, T289, R292, E293, E294, Q295, Y296, N297,S298, T299, Y300, R301, V303, V305, K317, K320, S324, K326, A327, P329,A330, I332, E333, K334, T335, S337, A339, Q342, R344, R355, T359, L398,S375, S383, N384, Q386, N389, K392, F404, G420, K340, Q438, N421, andY436, where the letter denotes a specific amino acid and the numberdenotes the position of the particular amino acid. Even more preferredwere variants including those with a non-natural amino acid replacingpositions S239, F241, K246, S267, H268, E269, D270, P271, E272, K274,F275, D280, G281, V282, E283, N286, T289, R292, E293, E294, Q295, Y296,N297, S298, T299, Y300, R301, V303, V305, K317, K320, S324, K326, A327,P329, A330, I332, E333, K334, T335, S337, A339, Q342, R344, R355, T359,L398, S375, Q386, N389, K392, F404, G420, K340, Q438, and N421. Stillmore preferred were variants including those with a non-natural aminoacid replacing positions S239, F241, S267, E269, D270, P271, E272, V282,N286, R292, E293, Y296, S298, P329, A330, K334, T335, K340, Q342, R355,T359, Q386, N389, F404, G420, N421, and Q438. Variants with anon-natural amino acid replacing positions S239, E293, K334, Q342, R355,T359, and N389 were identified as particularly preferred.

It was also noted that the expression, suppression efficiency,conjugation efficiency, cell-binding, and cell killing were notpredictable based on the published crystal structure of Fc. For example,E293 and K334 would not be predicted to accept substitution with anaromatic phenylalanine derivative based on their positions within the Fcstructure, yet both express well and are potent killers, with IC₅₀values of 2.7 and 3.0 ng/ml, respectively. Conversely, S131 waspredicted to be an excellent position for conjugation based on itsavailability on the surface of the heavy chain, yet derivatives withsubstitutions in this position were poor conjugators (DAR of 0.65) andpoor killers (IC₅₀ of 27.1 ng/mL). Thus, it was concluded thatexperimentation was required to identify optimal sites for expression,suppression, conjugation, and cell killing.

It should also be noted that sites near the N-linked glycosylation siteof trastuzumab, N297, were found to be well-suited for conjugation. Inparticular, sites from R292 through R301, V303 and V305 were found toexhibit desirable cell-killing and/or conjugation properties. It waspossible to assess these sites because of the aglycosylated form of thetrastuzumab variants produced in the cell-free protein synthesisreaction as described above.

Example 3 Fidelity of Incorporation of a Non-Natural Amino Acid in anExemplary Antibody-Drug Conjugate

This Example describes an evaluation of the fidelity of incorporation ofa non-natural amino acid in an exemplary antibody-drug conjugate.Trastuzumab substituted with p-azido-phenylalanine substituted at S136conjugated with DBCO-MMAF was digested with trypsin and analyzed on anAgilent 6520 Accurate Mass Q-TOF LC-MS system equipped with anano-electrospray ChipCube source. Peptides containing potentialmisincorporated amino acids and their wild-type counter parts weresearched using extracted ion chromatograms from the LC-MS data within±10 ppm of the theoretical m/z for the z=1-4 charge states of thepeptide. If the m/z of the found peak were within ±10 ppm of thetheoretical m/z and the correct charge state they were considered to bepotential matches until MS/MS verification. In the absence of signalfrom misincorporation, the signal was assumed to be equal or less thanthe noise in the mass spectrum. The minimum fidelity rate was calculatedas a noise to signal ratio. The signal was measured as the average ofall monoisotopic ions across the peptide's elution profile for the mostabundant charge state and the noise was estimated in the same spectrumin a region adjacent to the exhibiting a minimum amount of signal. Wherean observed peptide contained a misincorporation event was found, itsaveraged signal was used in place of the noise measurement.

In the peptide map of the tested antibody-drug conjugate, peptide withnnAA+ conjugated drug was detected at charge states 2 and 3 with anerror of less than 4 ppm. At position 136, no tyrosine incorporation waspresent and the incorporation efficacy for pAzPhe was determined to be98.3%. No tyrosine miss-incorporation was detected at the Phe positionsacross the detected IgG sequence. The overall, incorporation efficiencyfor Phe was measured to be at minimum 99.7%.

TABLE 1 IC₅₀ ¹⁴C SKBR3 IMAC expression CFPS cell gel rank ¹⁴CSuppression Cell HIC killing Variant μg/mL order μg/mL efficiencyBinding DAR Profile (ng/ml) S136 151 77 35% + 1.54 WR 7.9 S239 161 2 12657% + ND Single 5.2 Peak A118 156 7 105 48% + 1.51 WR 6.5 K246 162 18 7534% + ND Single 6.6 Peak S119 191 6 109 50% + 1.41 7.4 S132 307 1 15872% + 1.58 WR 8 A162 145 17 77 35% + 1.38 NWR 8 V005 186 3 124 56% +0.99 NWR 11.5 S191 183 4 114 52% + 0.68 WR 13.9 S074 177 9 96 44% + 0.78NWR 9.9 T135 151 8 96 44% + 1.03 NWR 10.1 A084 150 5 114 52% + 1.28 WR 9G194 145 11 89 40% + 1.1 WR 10.8 T139 138 24 55 25% + 1 WR 9.6 A172 13021 64 29% + 1.3 NWR 12.5 S134 124 16 80 36% + 1.37 WR 8.9 G137 112 15 8338% + 1.23 WR 9.3 A023 110 19 70 32% + 1.33 NWR 9.8 S165 103 20 68 31% +1.04 NWR 9.5 F241 99 13 87 39% + ND Single 8.4 Peak S160 96 14 86 39% +1.34 WR 10 P238 93 22 60 27% + 0.47 NWR 12.9 T155 89 10 95 43% + 1.19 WR10.5 V264 88 32 29 13% + ND Single 10.5 Peak S176 74 31 38 17% + 0.82NWR 12.4 G138 72 29 43 20% + 1.16 WR 11.2 G065 68 30 40 18% + ND NWR 9.6G042 68 26 49 22% + 1.12 NWR 13.8 F243 67 22 60 27% + 0.12 NWR 10.4 V26247 36 18 8% shifted rt ND NWR 8.2 D265 56 38 13 6% + ND Single 9 PeakL174 52 28 43 20% + 1.07 NWR 12.8 S219 26 27 45 20% + 1.18 NWR 13.6 S13176 25 49 22% 0.65 27.1 G161 43 33 26 12% 0.97 NWR 87.7 T164 17 35 22 10%0.68 NWR 17.5 T195 26 39 14 6% 1.1 NWR S177 23 34 24 11% + 0.68 NWR 13.6

TABLE 2 ¹⁴C IC₅₀ IMAC expression CFPS Cell Gel rank ¹⁴C Suppression HICKilling Variant (ug/ml) order μg/mL Efficiency Binding DAR Profile(ng/ml) E293 147.6 9 50.7 19% + 1.5  WR 2.7 K334 135.7 3 72.5 28% + 0.12Single 3 Peak E269 91.2 24 25.3 10% + 1.21 WR 1.4 S298 76.9 34 10.6 4% +1.17 NWR 1.9 R292 160.9 7 54.8 21% + ND Single 2.4 Peak with sh E272111.2 19 32.2 12% + 1.05 WR 3 V282 117.4 15 39.7 15% + 0.62 NWR 3.1 Y296142.7 14 39.9 15% + ND Single 3.5 Peak D270 122.6 12 43.8 17% + NDSingle 3.9 Peak N286 89.8 20 31.1 12% + 0.71 NWR 4 E333 96.1 8 52.620% + 0.55 NWR 5 S324 88.5 5 59.7 23% + 0.3  NWR 5.8 H268 140.2 4 67.626% + 0.46 NWR 7.9 T289 128.2 11 44 17% + 0.26 NWR 8.2 F275 97.3 13 42.416% + 0.24 NWR 9 I332 98.1 6 54.9 21% + 0.88 NWR 10 T335 123.7 2 82.432% + 0.33 NWR 11.2 P329 61.4 16 36.9 14% + 1.05 NWR 3.6 P271 78.2 1832.3 12% + 1.05 WR 4.4 A330 63.6 10 44.7 17% + 1.51 NWR 4.5 Y300 124.027 17.4 7% + 0.13 NWR 4.8 S267 22.1 29 12.4 5% + 1.57 NWR 5.1 A327 38.022 26.3 10% + 0.69 NWR 5.2 G281 31.0 32 11.5 4% + 0.27 NWR 5.2 V305 42.835 10.2 4% + 0.06 NWR 5.5 N297 34.0 37 7.8 3% + 0.09 NWR 6.5 Q295 35.628 14.2 5% + ND Single 6.8 Peak R301 41.8 39 6 2% + ND Single 7.8 PeakE294 27.9 30 12 5% + 0.65 NWR 8.3 E283 32.1 31 11.7 4% + 0.24 NWR 8.7D280 18.0 36 10.1 4% + ND Single 8.8 NWR K326 35.0 25 24.1 9% + 0.54 NWR9 K317 38.0 33 11.4 4% + 0.22 NWR 9.5 K274 36.3 26 19.3 7% + 0.45 WR10.2 T299 20.4 40 4.5 2% + ND NWR 10.3 V303 93.6 21 29 11% + 0.01 NWR11.1 K320 13.5 38 7.1 3% + 0.93 NWR 11.9 Y278 181.6 1 94 36% + ND WR22.5

TABLE 3 IC₅₀ IMAC ¹⁴C CFPS cell gel expression ¹⁴C Suppression HICkilling Variant μg/mL rank order μg/mL Efficiency Binding DAR Profile(ng/ml) R355 86 7 36 21% + 1.14 WR 13.8 T359 84 4 39 23% + 1.13 WR 7.5N389 63 6 37 22% + 1.54 WR 9.7 S337 56 5 39 23% + ND Unresolved 6.9 A33994 2 45 26% + ND Unresolved 7.7 L398 49 11 24 14% + ND Single 9.4 PeakQ438 56 36 ND ND + 1.46 WR 10 N421 27 15 18 11% + 1.35 WR 10.5 S375 68 827 16% + ND Single 12.3 Peak R344 52 14 20 12% + 1.13 WR 16.7 G420 19 1815 9% + 1.34 WR 9.4 K340 20 20 12 7% + 1.49 Low 12.9 Signal Q386 9 19 138% + 1.49 NWR 16.9 K392 11 34 2 1% + 1 NWR 18 S383 39 13 23 13% + 0.72WR 21.3 M358 25 16 16 10% + 0.93 21.6 E356 44 10 25 14% + 0.66 WR 27K360 55 12 23 14% + 0.42 WR 31.2 Y436 22 35 ND ND + 0.6 Low 32.6 SignalN384 127 1 69 40% + 0.25 WR 34.9 Q342 6.5 30 4.5 3% + 1.59 Low 35.1Signal

Example 4 Assessment of the Thermal Stability of Exemplary Antibody-DrugConjugates

This example describes experiments designed to measure the thermalstability (T_(m)) of aglycosylated trastuzumab and trastuzumab variants.The thermal shift assay was carried out by mixing the protein to beassayed (Sutroceptin and variants) with an environmentally sensitive dye(SYPRO Orange, Life Technologies Cat #S-6650) in a buffered solution(PBS), and monitoring the fluorescence of the mixture in real time as itundergoes controlled thermal denaturation. The final concentration ofthe protein in the assay mixture was between 100-250 μg/mL, and the dyewas 1:1000 diluted from the original stock (Stock dye is 5000× in DMSO).After dispensing 5 μL aliquots of the protein-dye mixture in a 384-wellmicroplate (Bio-Rad Cat #MSP-3852), the plate was sealed with anoptically clear sealing film (Bio-Rad Cat #MSB-1001), and placed in a384-well plate real-time thermocycler (Bio-Rad CFX384 Real Time System).The protein-dye mixture was heated from 25° C. to 95° C., at incrementsof 0.1° C. per cycle (˜1.5° C. per minute), allowing 3 seconds ofequilibration at each temperature before taking a fluorescencemeasurement. At the end of the experiment, the melting temperature(T_(m)) was determined using the Bio-Rad CFX manager software. Forprotein samples with complex thermal transition profiles, the meltingtemperature (T_(m)) is calculated from the negative first-orderderivative plot of fluorescence intensity (Y-axis) against temperature(X-axis), or by fitting the data to the Boltzmann sigmoidal model. Thedifference in melting temperature of IgG variants compared to thewild-type protein is a measure of the thermal shift for the proteinbeing assayed.

The results of this assay for certain variants are shown in Table 4. Ingeneral, deflections in T_(m) significantly below unsubstitutedtrastuzumab, particularly in T_(m)1, indicate an undesirable loss ofstability and/or a propensity to aggregate. As such, trastuzumabvariants that exhibit a T_(m)1 and/or T_(m)2 within about 5° C. ofunsubstituted trastuzumab are preferred. More preferred are thosevariants that exhibit a T_(m)1 and/or T_(m)2 within about 3° C. ofunsubstituted trastuzumab. Still more preferred are those variants thatexhibit a T_(m)1 and/or T_(m)2 within about 2° C. of unsubstitutedtrastuzumab. Still more preferred are those variants that exhibit aT_(m)1 and/or T_(m)2 within about 1° C. of unsubstituted trastuzumab.The T_(m)1 and T_(m)2 can be measured in the unconjugated or conjugatedforms.

TABLE 4 Antibody or Antibody-Drug Trastuzumab Conjugate Variant T_(m)1(° C.) T_(m)2 (° C.) Antibody Aglycosylated 63.5 +/− 0.6 76.5 +/− 0.1trastuzumab Antibody T359 64.0 +/− 0.4 76.8 +/− 0.0 Antibody E293 59.7+/− 0.7 76.1 +/− 0.1 Antibody K334 49.5 +/− 0.8 76.5 +/− 0.1 AntibodyR355 63.6 +/− 0.4 76.8 +/− 0.1 Antibody N389 62.8 +/− 0.1 76.8 +/− 0.0Antibody-Drug T359 63.9 +/− 0.6 76.7 +/− 0.1 conjugate Antibody-DrugE293 59.2 +/− 0.0 76.0 +/− 0.3 conjugate Antibody-Drug K334 59.0 +/− 0.276.3 +/− 0.2 conjugate Antibody-Drug R355 63.7 +/− 0.4 76.6 +/− 0.1conjugate Antibody-Drug N389 61.9 +/− 0.5 76.4 +/− 0.0 conjugate

Example 5 Preparation and Conjugation of Exemplary Fc Conjugates

This example describes preparation of exemplary Fc conjugates that areconjugated to a fluorescent dye. As shown in the example, certain testedresidues that conjugated well to the dye also conjugated well to acytotoxic agent in the context of Fc present in a full-lengthaglycosylated antibody. As such, it is believed that residues that aregood sites for conjugation in a full length antibody are also good sitesfor conjugation to Fc.

To make the Fc conjugates, site directed mutagenesis was performed usinga pYD plasmid containing coding region of Fc portion of aglycosylatedtrastuzumab with C-terminus his as DNA template and syntheticoligonucleotides containing mutations of interest in both sense andantisense directions. Oligonucleotides of each mutation were added toDNA template and PHUSION® polymerase (Thermo, Cat#F53 is) to a finalvolume of 20 μL. The final concentration of each component was 0.16 μMof each oligonucleotide, 0.5 ng/μL template DNA, 0.02 U/μL PHUSION®polymerase in HF buffer (Thermo) containing 1.5 mM MgCl₂ and 200 μMdNTP. Mixture was incubated at 98° C. 5 m, 18 PCR cycles (98° C. 30 s,55° C. 1 m, 72° C. 4 m), 10 m at 72° C. and stored at 4° C. DpnI (NEB)was added to the mixture to final concentration of 0.6 U/μL andincubated for 37° C. 1 h. 5 μL of each mixture was transformed into 50μL of Chemically Competent E. coli cells according to manufacturesprocedure (Invitrogen, MultiShot™ 96-Well Plate TOP10). Transformedcells were recovered in 200 μL SOC(Invitrogen) 37° C. 1 h and platedonto Luria-Bertani (LB) agar supplemented with 50 μg/mL Kanamycin(Teknova). After 37° C. 24 h, colonies were picked using Qpix2 (Genetix)into 200 μL LB with 7.5% glycerol and 50 μg/mL Kanamycin, and grown at37° C. for 24 h, 20 μL of culture was used for rolling circleamplification and sequenced by primer extension using T7 (SEQ ID NO: 2,5′TAATACGACTCACTATAGG 3′) and T7 term (SEQ ID NO: 3, 5′GCTAGTTATTGCTCAGCG 3′) primers (Sequetech). Sequence was analyzed bySequencher (Gene Codes), and clones containing mutations were arrayedinto 96 well plates.

Expression of the Fc proteins was performed using cell free proteinsynthesis system as described in Zawada et al., 2011, Biotechnol.Bioeng. 108(7)1570-1578 with the modifications described below.Cell-free extracts were treated with 50 μM iodoacetamide for 30 min atRT (20° C.) and added to a premix containing all other components exceptfor DNA. Cell free reactions were initiated by addition of plasmid DNAof variants of interest and incubated at 30° C. for 5 h 850 rpm in 96well plates. Reaction was incubated further at 4° C. for 11 h. The finalconcentration of each components was 30% cell extract, 20 uM pAzF-tRNA,2 mM GSSG, 100 μg/mL E. coli DsbC, 8 mM magnesium glutamate, 10 mMammonium glutamate, 130 mM potassium glutamate, 35 mM sodium pyruvate,1.2 mM AMP, 0.86 mM each of GMP, UMP, and CMP, 2 mM amino acids (except0.5 mM for Tyrosine), 4 mM sodium oxalate, 1 mM putrescine, 1.5 mMspermidine, 15 mM potassium phosphate, 100 nM T7 RNAP, 10 μg/mL DNA ofeach Fc Variant.

Samples were then purified by IMAC Phytip and by Protein A Phytip. Forsamples prepared by IMAC Phytip, each Fc variant was produced in 30 μLscale in a 96 well plate. Each CFPS reaction was diluted with 30 μL PBS(Gibco, pH7.4). The mixture was centrifuged 5000×g 4° C. for 15 m.Supernatant was captured with IMAC Phytip containing 5 μL resin(PhyNexus) by pipetting up and down 4 times slowly with a flow rate of250 μl/min. Resin was washed with 200 μL IMAC binding buffer (50 mM TrispH 8.0, 300 mM NaCl, 10 mM imidazole) by pipetting up and down twice ata flow rate of 500 μL/min. This process was repeated once with anadditional 200 μL IMAC binding buffer. Bound protein was eluted with 125μL IMAC elution buffer (50 mM Tris pH 8.0, 300 mM NaCl, 500 mMImidazole) by pipetting up and down 4 times at a flow rate of 250μL/min.

For samples purified by Protein A Phytip, each Fc variant was producedin 30 μL scale in a 96 well plate. Each CFPS reaction was diluted with30 μL PBS (Gibco, pH7.4). The mixture was centrifuged 5000×g 4° C. for15 m. Supernatant was captured with Protein A Phytip containing 5 μLresin (PhyNexus) by pipetting up and down 4 times with a flow rate of250 μl/min. Resin was washed with 200 μL PBS by pipetting up and downtwice at a flow rate of 500 μL/min. Resin was then washed with 200 μL150 mM NaCl. Bound proteins was eluted with 125 μL Glycine at pH 3.0(100 mM) by pipetting up and down 4 times at a flow rate of 250 μL/min.The eluted protein was immediately neutralized with 50 μL of 1M Tris8.0. The neutralized elution buffer contains 71 mM Glycine and 285 mMTris with pH of approximately 7.8.

The conjugation to an exemplary fluorescent dye was performed asfollows. DIBO-TAMRA (Invitrogen C-10410) (structure shown as FIG. 5) wasdissolved in anhydrous DMSO to a final concentration of 5 mM. Thecompound was diluted 10× into with PBST (PBS with 0.2% Tween 20) to 500μM and then added to Fc-his variants in IMAC elution buffer to the finaldrug concentration of 50 μM. Mixture was incubated at 30° C. 250 rpm for16 h in a Thermomixer (Thermo). Sodium Azide was added to samples tofinal concentration of 100 mM.

To determine amounts of FC expression, Affinity Phytip purified sampleswere analyzed for protein A binding using ForteBio Protein A tips(18-5010). Each sample was diluted 2 to 10 fold in PBS based kineticbuffer (ForteBio 18-5032). Samples were incubated with ForteBio ProteinA tip for 300 s and dissociated for 600 s in kinetic buffer. On rate wasmeasured and compared to a standard curve generated using purifiedFc-his (0.4 to 25 ug/mL) to determine concentration. Expression levelswere normalized to expression of the S375pAzF variant.

To determine the amount of conjugation, IMAC or Protein A Phytippurified samples were mixed with equal volume of 2× Laemmli samplebuffer and fractionated by 4-12% gel (BioRad). Fluorescence intensity ofTAMRA conjugated protein was visualized by GelDoc EZ (BioRad, UVexcitation filter (280-400 nM)). Relative fluorescence intensity of eachgel band was quantitated and normalized to that of conjugated product ofFc variant S375pAzF produced on the same plate using correspondingplasmid DNA (SD2009).

Of tested Fc proteins, substitutions at positions corresponding to S239and Y296 were particularly well-expressed and conjugated. Accordingly,substitutions at these positions are particularly preferred for Fcconjugates.

Example 6 Assessment of Expression of HC-F404 in an RF-1 AttenuatedExtract

To initially assess the effects of using an RF-1 attenuated E. colistrain on incorporation of a non-natural amino acid, 12 variants whichpoorly expressed in the RF-1 positive strain were expressed and scaledup in the RF-1 attenuated strain. One such variant, HC-F404, exhibitedexceptionally desirable properties, as discussed below.

The cell free extract used for this cell free protein synthesis reactionwas prepared from an OmpT sensitive RF-1 attenuated E. coli strain whichhas also been engineered to produce an orthogonal CUA-encoding tRNA forinsertion of a non-natural amino acid at an Amber Stop Codon. Afteraddition of DNA template that encodes for HC-F404 and WT LC, the cellfree reaction was incubated at 30° C. for 12 h on a shaker at 650 rpm ata 1 ml scale ×6 (for a total of 9 ml) in a Flower plate (m2p-labs#MTP-48-B). The protein was then purified over Protein A and CaptoAdhere resin using a Protein Maker (Emerald Bio).

Conjugation with drug (DBCO-MMAF) and preparation of Antibody DrugConjugates (ADC's) for further analysis was done as describedpreviously.

Themofluor analysis of the variants was carried out as describedpreviously

HC variant F404 generated both in during TAG scan as well as in theintermediate scale showed good cell-killing capability (TAG Scan I:IC50=0.018 nM; Intermediate Scale up: IC50=0.023 nM), but DAR estimationon the basis of its HIC profile proved problematic, due to lowresolution of the peaks, possibly due to inaccessibility of the drug tothe analytical column during HIC analysis. However, MS analysis showedthat it had a DAR of 1.74. Also, thermofluor analysis showed that theADC version of HC-F404 had an improved (higher thermal stability) T_(m)1(increase of 2.2° C.) compared to the antibody only.

TABLE 5 Properties of HC-F404 variant Final Purified SKBR3 Cell ADCconcentration MS Killing IC50 Chain Variant (ug/mL) DAR Profile (ng/mL)HC F404 541 1.74 WR* 3.5 *This DAR result is based on LC-MS analysis.The conjugated variant produced a Not-Well-Resolved (NWR) peak on theHIC assay, and DAR was determined using LC-MS

TABLE 6 Thermofluor results for HC-F404 variant Trastu- zumab AntibodyOnly Antibody-Drug Conjugate Chain Variant T_(m)1 (° C.) T_(m)2 (° C.)T_(m)1 (° C.) T_(m)2 (° C.) HC F404 61.2 +/− 0.2 76.6 +/− 0.1 63.5 +/−0.4 76.5 +/− 0.1

Example 7 Characterization of Exemplary Antibody-Drug Conjugates:Release Factor Analysis

TAG Scan I Methods and List of Selected Variants

The cell free extract used for cell free protein synthesis reactionswere prepared from an OmpT sensitive RF-1 attenuated E. coli strainwhich has also been engineered to express an orthogonal CUA-encodingtRNA for insertion of a non-natural amino acid at an Amber Stop Codon.After addition of DNA template, cell free reactions were incubated at30° C. for 12 h on a shaker at 650 rpm in Flower plates (m2p-labs#MTP-48-B). For synthesis of light chain variants (variants which hadnnAA incorporation on the light chain), a DNA ratio of 4 ug/mL of lightchain DNA to 8 μg/mL of heavy chain DNA was used. Each trastuzumabvariant was produced in 1 mL scale in 48-well flower plates insinglicate. A total of 6 plates were used to express the variants.

Supernatant was captured with IMAC Phytip containing 40 μL resin bypipetting up and down 10 times slowly at a flow rate of 4.2 uL/min.Bound protein was eluted with 125 μL IMAC elution buffer (50 mM Tris pH8.0, 300 mM NaCl, 500 mM Imidazole). Following purification, IMACpurified variants were quantified on a Caliper GXII system by comparingwith by mass standards of HERCEPTIN® run on the same Protein ExpressLabChip (Caliper LifeSciences #760499). Samples were prepared foranalysis as specified in the Protein Express Reagent Kit (Caliper LifeSciences #760328) with the exception that the samples (mixed in samplebuffer) were heated at 65° C. for 10 minutes prior to analysis on theCaliper system.

Conjugation with drug (DBCO-MMAF) and preparation of Antibody DrugConjugates (ADC's) for further analysis was done as describedpreviously.

Positive results of these characterization experiments are presented inTable 7. In the Table, the following properties of the variants areshown: final concentration of antibody-drug conjugate made, ability tobind HER2-expressing cells, drug-antibody ratio (DAR, expressed as thenumber of cytotoxic agents per antibody), a comment regarding the HICprofile, and the observed IC₅₀ from the cell-killing assay describedpreviously. In the comments regarding the HIC assay, SP corresponds to aprofile that resolved well as a single peak, NWR corresponds to a poorlyresolved profile and WR corresponds to a well-resolved HIC profile.Several of the variants that exhibited a single peak profile alsoexhibited potent killing, which indicates that the variant was indeedconjugated with drug, but failed to resolve on the HIC column.

TABLE 7 Selected Variants for Tag Scan I Final SKBR3 cell Purified ADCHIC killing IC₅₀ Variants Chain (ug/mL) DAR Profile (ng/mL) V282 HC 521.0 NWR 1.6 T289 HC 46 0.3 WR 2.1 Y296 HC 32 1.5 WR 7.2 A330 HC 80 ND SP2.4 T335 HC 43 0.7 NWR 2.3 N361 HC 42 1.0 WR 5.9 S400 HC 51 0.5 WR 7.1F404 HC 20 ND NWR 4.3 V422 HC 100 1.3 WR 4.0 S440 HC 74 1.2 WR 6.1 T260HC 37 0.1 NWR 4.4 S267 HC 94 1.7 WR 7.2 H268 HC 45 0.4 NWR 7.5 E272 HC50 1.2 WR 3.0 K274 HC 64 0.8 WR 2.8 R292 HC 101 ND SP 9.0 E293 HC 1331.7 WR 9.0 N297 HC 45 1.7 WR 4.7 S298 HC 60 1.5 WR 5.4 V303 HC 75 1.5 WR12.7 V305 HC 66 1.5 WR 7.0 I332 HC 47 ND NWR 2.6 E333 HC 47 ND NWR 2.6K334 HC 83 1.8 WR 6.9 K340 HC 76 1.5 WR 4.4 G341 HC 29 1.5 WR 3.8 Q342HC 60 1.3 WR 4.8 P343 HC 51 0.8 WR 6.6 R355 HC 54 1.4 WR 4.6 Q362 HC 891.1 WR 6.3 Q386 HC 124 ND NWR 4.0 K392 HC 40 1.6 WR 3.4 S424 HC 24 1.3WR 4.9 Q438 HC 33 1.5 WR 5.2 S442 HC 57 1.4 WR 4.3 L443 HC 39 1.4 WR 3.0

Preferred variants include those with a non-natural amino acid replacingthese positions of the heavy chain (HC): V282, T289, Y296, A330, T335,N361, S400, F404, V422, S440, T260, S267, H268, E272, K274, R292, E293,N297, S298, V303, V305, I332, E333, K334, K340, G341, Q342, P343, R355,Q362, Q386, K392, F404, S424, Q438, S442 and L443.

Variants that have a DAR of at least about 0.7 are also preferred.Preferred variants include those with a non-natural amino acid replacingthese positions of the heavy chain (HC): V282, Y296, T335, N361, V422,S440, S267, E272, K274, E293, N297, S298, V303, V305, K334, K340, G341,Q342, P343, R355, Q362, K392, F404, S424, Q438, S442 and L443.

Variants that have a DAR of at least about 1.0 are also preferred. Stillmore preferred were variants including those with a non-natural aminoacid replacing these positions of the heavy chain: V282, Y296, N361,V422, S440, S267, E272, E293, N297, S298, V303, V305, K334, K340, G341,Q342, R355, Q362, K392, F404, S424, Q438, S442 and L443.

Variants that have a DAR of at least about 1.2 are also preferred. Stillmore preferred were variants including those with a non-natural aminoacid replacing these positions of the heavy chain: V282, Y296, V422,S440, S267, E272, E293, N297, S298, V303, V305, K334, K340, G341, Q342,R355, K392, F404, S424, Q438, S442 and L443.

Variants that have a DAR of at least about 1.5 are also preferred. Stillmore preferred were variants including those with a non-natural aminoacid replacing these positions of the heavy chain: V282, Y296, S267,E293, N297, S298, V303, V305, K334, K340, G341, K392, F404, and Q438.

Example 8 Characterization of Exemplary Antibody-Drug Conjugates:Additional Release Factor Analysis

TAG Scan II Methods and List of Selected Variants

The cell free extract used for cell free protein synthesis reactionswere prepared from an OmpT sensitive RF-1 attenuated E. coli strainwhich has also been engineered to produce an orthogonal CUA-encodingtRNA for insertion of a non-natural amino acid (p-azido-phenylalanine)at an Amber Stop Codon. After addition of DNA template, cell freereactions were incubated at 30° C. for 12 h on a shaker at 650 rpm inFlower plates (m2p-labs #MTP-48-B). For synthesis of light chainvariants (variants which had nnAA incorporation on the light chain), aDNA ratio of 4 μg/mL of light chain DNA to 8 μg/mL of heavy chain DNAwas used. Each trastuzumab variant was produced in 1 mL scale in 48-wellflower plates in singlicate. A total of 6 plates were used to expressthe variants.

Supernatant was captured with IMAC Phytip containing 40 μL resin bypipetting up and down 10 times slowly at a flow rate of 4.2 μL/min.Bound protein was eluted with 125 μL IMAC elution buffer (50 mM Tris pH8.0, 300 mM NaCl, 500 mM Imidazole). Following purification, IMACpurified variants were quantified on a Caliper GXII system by comparingwith by mass standards of HERCEPTIN® run on the same Protein ExpressLabChip (Caliper LifeSciences #760499). Samples were prepared foranalysis as specified in the Protein Express Reagent Kit (Caliper LifeSciences #760328) with the exception that the samples (mixed in samplebuffer) were heated at 65° C. for 10 minutes prior to analysis on theCaliper system.

Conjugation with drug (DBCO-MMAF) and preparation of Antibody DrugConjugates (ADCs) for further analysis was done as described previously.

Positive results of these characterization experiments are presented inTable 8. In the Table, the following properties of the variants areshown: final concentration of antibody-drug-conjugate made, theefficiency of expression of full length product compared to wild-type(suppression efficiency), ability to bind HER2-expressing cells,drug-antibody ratio (DAR, expressed as the number of cytotoxic agentsper antibody), a comment regarding the HIC profile, and the observedIC₅₀ from the cell-killing assay described previously. In the commentsregarding the HIC assay, SP corresponds to a profile that resolved wellas a single peak, NWR corresponds to a poorly resolved profile and WRcorresponds to a well-resolved HIC profile. Several of the variants thatexhibited a single peak profile also exhibited potent killing, whichindicates that the variant was indeed conjugated with drug, but failedto resolve on the HIC column.

In total, trastuzumab variants having a non-natural amino acid at 269sites that were screened for the activities as described. Table 8presents the 71 variants that had desirable DAR, expression profiles,suppression efficiencies, and/or IC₅₀ values. None of these propertiescould be predicted prior to testing, and many times the values for eachindividual property varied unpredictably within a sample. For example,one would generally expect that variants with high DAR would exhibitlower IC₅₀ values than those with low DAR. Nonetheless, the I51 variant,for example, exhibited a relatively low DAR of 0.6 yet exhibited an IC₅₀of 1 ng/ml.

TABLE 8 Tag Scan II Variants MMAF SKBR3, Suppression, (Post MQ) HICIC₅₀, % of wt by Variants Chain (ug/mL) DAR Profile ng/mL ¹⁴C D221 HC178 1.4 WR 9.6 75 K222 HC 194 1.5 WR 8.4 87 T225 HC 156 1.4 WR 8.3 81P227 HC 96 1.5 NWR 6.2 68 P230 HC 157 1.5 NWR 6.0 122 A231 HC 147 1.6NWR 5.5 97 P232 HC 107 1.5 NWR 5.5 86 G236 HC 74 1.6 WR 4.5 70

Preferred variants include those with a non-natural amino acid replacingthese positions of the heavy chain (HC): D221, K222, T225, P227, P230,A231, P232, and G236.

Variants that have a DAR of at least about 0.7 are also preferred.Preferred variants include those with a non-natural amino acid replacingthese positions of the heavy chain (HC): D221, K222, T225, P227, P230,A231, P232, and G236.

Variants that have a DAR of at least about 1.0 are also preferred. Stillmore preferred were variants including those with a non-natural aminoacid replacing these positions of the heavy chain: D221, K222, T225,P227, P230, A231, P232, and G236.

Variants that have a DAR of at least about 1.2 are also preferred. Stillmore preferred were variants including those with a non-natural aminoacid replacing these positions of the heavy chain: D221, K222, T225,P227, P230, A231, P232, and G236.

Variants that have a DAR of at least about 1.5 are also preferred. Stillmore preferred were variants including those with a non-natural aminoacid replacing these positions of the heavy chain: K222, P227, P230,A231, P232, and G236.

Example 9 Characterization of Exemplary Antibody-Drug Conjugates: Scaleup Analysis

Scale Up of Select Variants from TAG II Scan

Based on DAR and cell-killing data, a subset of trastuzumab variantswith desirable characteristics were picked for small scale cell freeexpression in order to generate material for further characterization.The heavy chain variants selected for small scale production were: D221and K222.

The cell free reaction mix in which the variants were synthesizedcomprised of a 80%:20% blend of cell free extracts made from an OmpTsensitive RF-1 attenuated E. coli strain, and an OmpT sensitive RF-1attenuated E. coli strain that was engineered to express an orthogonalCUA encoding tRNA, respectively. All variants were scaled up to 9 ml inflower plates (1.5 mL×6 replicates) and purified using Protein Maker(Emerald Bio).

Conjugation with drug (DBCO-MMAF) and preparation of Antibody DrugConjugates (ADC's) for further analysis was done as describedpreviously.

Positive results of these characterization experiments are presented inTable 9. In the Table, the following properties of the variants areshown: final concentration of antibody-drug-conjugate made, theefficiency of expression of full length product compared to wild-type(suppression efficiency), ability to bind HER2-expressing cells,drug-antibody ratio (DAR, expressed as the number of cytotoxic agentsper antibody), a comment regarding the HIC profile, and the observedIC₅₀ from the cell-killing assay described previously. In the commentsregarding the HIC assay, SP corresponds to a profile that resolved wellas a single peak, NWR corresponds to a poorly resolved profile and WRcorresponds to a well-resolved HIC profile.

TABLE 9 A Subset of Preferred Fc Variants Final Purified SKBR3 Cell ADCconcentration Killing IC₅₀ Chain Variant (ug/mL) DAR HIC Profile (ng/mL)HC D221 284 1.16 WR 3.5 HC K222 308 1.21 WR 3.0

Example 10 Characterization of Exemplary Antibody-Drug Conjugates:Thermofluor Analysis

Thermofluor analysis of the variants was carried out as describedpreviously. Thermofluor results are in Table 10.

TABLE 10 Thermofluor data for small Scale production variantsTrastuzumab Antibody Only Antibody-Drug Conjugate Chain Variant T_(m)1(° C.) T_(m)2 (° C.) T_(m)1 (° C.) T_(m)2 (° C.) Aglycosylated 61.4 +/−0.6 76.2 +/− 0.1 N/A N/A Trastuzumab HC D221 59.1 +/− 0 76.7 +/− 0.1 59+/− 0.1 76.7 +/− 0.1 HC K222 59.1 +/− 0.1 76.7 +/− 0.1 59 +/− 0.1 76.5+/− 0.1

Variants subject to thermofluor analysis included those with anon-natural amino acid replacing these positions of the heavy chain:D221 and K222.

Example 11 Characterization of Exemplary Antibody-Drug Conjugates:Kinectics Analysis

Conjugation Kinetics of Selected Variants

Rates of conjugation were determined for five variants that showed widerange of drug to antibody conjugate ratio. Reactions were initiated bymixture of variants with drug (DBCO-MMAF) in PBS (pH7.4) at 20° C. induplicates for 15 min, 30 min, 1 hr, 2 hr, 4 hr, 8 hr and 16 hr. Finalconcentrations of antibody in mixture range from 0.2 to 2 uM. Final drugconcentration for all reaction is 100 uM. At the end of incubationperiod, NaAzide was added to the mixture to a final concentration of 10mM. Final mixture was purified by Zeba and MustangQ plates as previouslydescribed. Concentration of IgG was determined by Caliper usingHerceptin®as mass standard. Fraction of fully conjugated variants wasdetermined by HIC as previously described.

TABLE 11 Half-lives and DAR of variants HC-S112 HC-T110 HC-T77 HC-Y79HC-F126 T_(1/2) 6.2 h 10.5 h ~40 h ~270 h ND DAR 1.7 1.6 1.1 0.4 0

FIGS. 5A and 5B provides the HIC traces of two variants (HC T110 and HCS112). The traces showed three peaks (P0, P1 and P2) corresponding topeaks with unconjugated IgG, singly conjugated IgG and fully conjugateIgG, respectively.

FIGS. 6A through 6E show that conjugation of para-azido phenylalanine(pAzF) variants is site specific. Percentages of total of single andfully conjugated antibodies were separated by HIC.

Example 12 Characterization of Exemplary Antibody-Drug Conjugates:Suppression Comparison Analysis Comparison of Suppression by para-azidophenylalanine (pAzF) and par-azido methyl phenylalanine (pAzMeF)

Eleven variants with a range of suppression efficiencies were expressedin a cell-free protein synthesis reaction as follows as described inZawada et al., 2011, Biotechnol. Bioeng. 108(7): 1570-1578 with themodifications described below. Unsubstituted trastuzumab was also madeas a control. Cell-free extracts containing tRNA and OmpT sensitive RF1(80/20 blend of strains 16/23) were treated with 50 μM iodoacetamide for30 min at RT (20° C.) and added to a premix containing all othercomponents except for GSSG, non-natural amino acids, tRNA synthetases,T7 RNAP, DsbC, PDI, and template DNA. All remaining reagents except DNAwere then added to the mixture. Cell free reactions were initiated byaddition of plasmid DNA of selected variants and incubated at 30° C. for12 h on a shaker at 450 rpm in 96-well plates. The reaction wasincubated further at 4° C. for 5 h. The final concentration in theprotein synthesis reaction was 30% cell extract, 1 mM para-azidophenylalanine (pAzF) (RSP Amino Acids) with 0.37 mg/mL M. jannaschiipAzF-specific amino-acyl tRNA synthetase (FRS), or 1 mM para-azidomethyl phenylalanine (pAzMeF) with 0.37 mg/mL p-cyanophenylalaninespecific aminoacyl-tRNA synthetase (Young et al, 2011, Biochem. 50:1894-1900), 2 mM GSSG, 0.29 mg/mL PDI (Mclab), 100 μg/mL E. coli DsbC, 8mM magnesium glutamate, 10 mM ammonium glutamate, 130 mM potassiumglutamate, 35 mM sodium pyruvate, 1.2 mM AMP, 0.86 mM each of GMP, UMP,and CMP, 2 mM amino acids (except 0.5 mM for Tyrosine andPhenylalanine), 4 mM sodium oxalate, 1 mM putrescine, 1.5 mM spermidine,15 mM potassium phosphate, 100 nM T7 RNAP, 2 μg/mL trastuzumab lightchain DNA, 8 μg/mL trastuzumab-(His)₆ heavy chain DNA. Each trastuzamabvariant was produced in 100 uL scale in 96-well plates in duplicateswith ¹⁴C for each variant. It should be noted that all trastuzumabvariants thus produced were aglycosylated.

To monitor protein synthesis reactions were spiked with 3% (v/v)1-[U-¹⁴C]-leucine (300 mCi/mmole; GE Life Sciences, Piscataway, N.J.).The suppression of amber codon at different sites of the heavy chain andlight chain was determined by [¹⁴C]-autoradiograhy of reducing SDS-PAGEgels. Full length trastuzumab heavy chain and suppressed tastuzumabheavy chain variants run at 50 kD on SDS-PAGE. Full length trastuzumablight chain and suppressed tastuzumab light chain variants run at 30 kDon SDS-PAGE. Non suppressed (truncated) trastuzumab variants run at alower molecular weight. Amber suppression in the heavy or light chain isdetermined by:

${suppression} = \frac{{band}\mspace{14mu}{intensity}\mspace{14mu}{of}\mspace{14mu}{suppressed}\mspace{14mu}{heavy}\mspace{14mu}{or}\mspace{14mu}{light}\mspace{14mu}{chain}\mspace{14mu}{TAG}\mspace{14mu}{variant}}{{band}\mspace{14mu}{intensity}\mspace{14mu}{of}\mspace{14mu}{wild}\mspace{14mu}{type}\mspace{14mu}{heavy}\mspace{14mu}{or}\mspace{14mu}{light}\mspace{14mu}{chain}}$

Band intensity was determined by ImageQuant (Amersham Biosciences Corp.;Piscataway, N.J.). In FIG. 6A, the suppression efficiency of pAzF andpAzMeF variants are compared. Suppression efficiency is calculated asvariant HC band intensity over wild type HC band intensity or variant LCband intensity over wild type LC band intensity. In FIG. 6B, the solubleIgG yield of pAzF and pAzMeF variants are compared. The amount of IgG iscomputed according to the formula: soluble counts/full counts*IgG bandintensity/total lane intensity*2* 74008.02 Da/47 Leucines.

Example 13 Transfer of Sites of Non-Natural Amino Acid Incorporation toAnother Antibody

To test whether sites for incorporation of non-natural amino acids canbe transferred between two distinct IgG sequences with predictable DARs,a second antibody containing a non-natural amino acid at representativesites was assessed. For this experiment, brentuximab was chosen as thesecond antibody (See SEQ ID NO: 3 and SEQ ID NO: 4). Several sites werechosen from the trastuzumab mutagenesis study exhibiting varying DARs.In the heavy chain, sites corresponded to K121, Y180, K133, S157, F126,and P127 (EU Numbering). In the light chain, sites corresponded to R142,N152, Q147, E161, K149, and Q155 (EU Numbering). The TAG stop codon wasinserted into the brentuximab sequence using standard quick change basedsite directed mutagenesis, and the identity of each variant wasconfirmed by DNA sequencing. Mini Prep DNA was prepared using a QiagenKit, for use as a template to drive the cell free protein synthesisreaction.

The variants were synthesized in a reaction mix that contained an OmpTsensitive RF-1 protein and an in vivo expressed orthogonal CUA encodingtRNA. All variants were scaled up to 9 ml in flower plates (1.5 mL×6replicates) and purified using a Protein Maker (Emerald Bio).

Conjugation with drug (DBCO-MMAF) and preparation of Antibody DrugConjugates (ADC's) for further analysis was done as describedpreviously. DARs were identified by the HIC profiling method previouslydescribed.

The results from the experiment suggest that sites can generally bepredictably transferred between two distinct IgGs.

TABLE 12A A Subset of Preferred HC variants DAR Variants trastuzumabbrentuximab HC-K121^(TAG) 1.6 1.4 HC-Y180^(TAG) 1.6 0.5 HC-K133^(TAG)0.7 0.8 HC-S157^(TAG) 0.9 1.1 HC-F126^(TAG) — — HC-P127^(TAG) — —

TABLE 12B A Subset of Preferred LC variants DAR Variants trastuzumabbrentuximab LC-R142^(TAG) 1.2 1.3 LC-N152^(TAG) 1.2 1.3 LC-Q147^(TAG)0.8 1 LC-E161^(TAG) 0.8 NA LC-K149^(TAG) 0.1 0.1 LC-Q155^(TAG) 0.1 0.1

To further characterize brentuximab conjugates, variants containingamber mutations at heavy chain positions K121 or F404 or light chainposition N152 were selected for scale up and additionalcharacterization. As a control, trastuzumab containing an amber mutationat F404 was also expressed expressed TAG codon was inserted byoverlapping PCR mutagenesis at the nucleotides corresponding topositions K121 and F404 on heavy chain and N152 on light chain andseparately cloned into expression vector pYD317.

The cell free reaction mix in which the brentuximab and trastuzumabvariants were synthesized comprised a blend of cell free extracts madefrom an OmpT sensitive RF-1 attenuated E. coli strain, and an OmpTsensitive RF-1 attenuated E. coli strain which was engineered to producean orthogonal CUA-encoding tRNA for insertion of a non-natural aminoacid at an Amber Stop Codon. The variants were expressed in a cell-freeprotein synthesis reaction as described in Zawada et al., 2011,Biotechnol. Bioeng. 108(7)1570-1578 with the modifications describedbelow. Cell-free extracts were treated with 50 μM iodoacetamide for 30min at RT (20° C.) and added to a premix containing all other componentsexcept for DNA encoding the variants of interest. The finalconcentration in the protein synthesis reaction was 30% cell extract, 1mM para-azido methyl phenylalanine (pAzMeF) (RSP Amino Acids), 0.37mg/mL M. jannaschii pAzMeF-specific amino-acyl tRNA synthetase (FRS), 2mM GSSG, 0.29 mg/mL PDI (Mclab), 30 μg/mL E. coli DsbC, 8 mM magnesiumglutamate, 10 mM ammonium glutamate, 130 mM potassium glutamate, 35 mMsodium pyruvate, 1.2 mM AMP, 0.86 mM each of GMP, UMP, and CMP, 2 mMamino acids (except 0.5 mM for Tyrosine and Phenylalanine), 4 mM sodiumoxalate, 1 mM putrescine, 1.5 mM spermidine, 15 mM potassium phosphate,100 nM T7 RNAP. Plasmid ratios of HC to LC were tested to find theoptimal condition: 3:1, 2:1, 1:1 and 1:2. Total plasmid concentrationkept constant at 10 ug/mL. After addition of DNA template, cell freereactions were incubated at 30° C. for 12 h on petri dishes and followedby 14C assay analysis. Maximum yield was achieved at 1:1 ratio. 10 mLcell-free reactions were carried under this condition.

To purify the variants, 10 ml of crude cell-free for each variant wasfirst diluted 1:0.5 with equilibration buffer (50 mM sodium phosphate,pH 7) and spun at 11,000×g for 30 minutes. The supernatant was thenpassed through a 0.45 micron syringe filter prior to being loaded with a2 minute residence time onto a pre-equilibrated 1 mL MabSelect SureHiTrap (GE Lifesciences) to capture the IgG variants. The column wasthen washed with 7.5 CV (column volume) of wash buffer 1 (100 mM sodiumphosphate and 800 mM Arginine, pH 7) and followed by 7.5 CV of washbuffer 2 (50 mM sodium phosphate and 0.5% (v/v) Triton X-100, pH 7.3).After washing with 7.5 CV of equilibration buffer, each variant waseluted with 4 CV elution buffer (100 mM sodium citrate and 300 mMArginine, pH 3). The elution pool was adjusted to pH 7 by addition of30% (v/v) of 1M Tris, pH9.

The collected elution pool was buffer exchanged into PBS via overnightdialysis in 10 kD Slide-A-Lyzer (Pierce) units. The dialyzed materialwas then concentrated using an Amicon Ultra-15 (Millipore) centrifugalfilter unit to a concentration of 5-10 mg/mL.

The purified variants were conjugated as follows. DBCO-MMAF 2 (ACMEBioscience; Palo Alto, Calif.) was dissolved in DMSO to a finalconcentration of 5 mM. The compound was diluted with PBS to aconcentration of 1 mM and then added to purified trastuzumab variants inIMAC elution buffer to achieve a final drug concentration of 100 μM.Mixture was incubated at RT (20° C.) for 17 hours. Reaction was stoppedby adding Sodium Azide to final concentration of 100 mM and bufferexchanged using Zeba plates (Thermo Scientific; Waltham, Mass.)equilibrated in 1×PBS.

DAR for the aglycosylated variants was determined by LC-MS as follows.ADCs by LCMS

Samples were run on a Waters Aquity UPLC system attached a Xevo QTOF.Proteins were separated on an Agilent PLRP-S column (2.3×50 mm, 5 μm,4000 Å) at 80° C. Mobile phases: A: 0.1% formic acid water; b: 20:80isopropanol:acetonitrile, 0.1% formic acid. Samples Were desalted oncolumn for 0.4 minutes at 10% B followed by a step gradient from 30% Bto 40% B over 7 minutes, 40% B to 60% B over 3 minutes. Data wasacquired over the whole LC elution using a cone voltage of 35V. Spectrawere analyzed using MassLynx software. DAR values were calculated as aweighted average and are shown in Table 13.

TABLE 13 DAR values for ADCETRIS ® ADCs Variant Drug DAR AglycosylatedBrentuximab IgG HC-F404 DBCO-MMAF2 1.87 Aglycosylated Brentuximab IgGHC-K121 DBCO-MMAF2 1.79 Aglycosylated Brentuximab IgG LC-N152 DBCO-MMAF21.74 Aglycosylated Trastuzumab IgG HC-F404 DBCO-MMAF2 1.97

Next, cell binding and cell killing activities of the variants weremeasured as follows. Karpas 299 and L540 cell lines were obtained fromGerman Collection of Microorganisms and Cell Cultures (DSMZ) and SKBR3and Raji cell lines were obtained from American Type Culture Collection(ATCC). Cells were grown in RPMI 1650 medium (Cellgro-Mediatech;Manassas, Va.) containing 20% heat-inactivated fetal bovine serum(Hyclone; Thermo Scientific; Waltham, Mass.), 2 mM glutamax (Invitrogen;Carlsbad, Calif.) and 1× penicillin/streptomycin (Cellgro-Mediatech;Manassas, Va.). Adherent SKBR3 cells were grown in DMEM/Nutrient F-12Ham (50:50) high glucose medium (Cellgro-Mediatech; Manassas, Va.)supplemented with 10% heat-inactivated fetal bovine serum, 2 mM glutamaxand 1× penicillin/streptomycin. All cells were grown and maintained at37° C. in a 5% CO2 incubator. SKBR3 were passaged by washing withcalcium/magnesium-free Phosphate Balanced Saline (PBS) and harvestedwith HyQTase (Hyclone; Thermo Scientific; Waltham, Mass.).

Cell killing activities of Trastuzumab and Brentuximab (ADCETRIS®)variants were measured using CellTiter-Glo™ cell proliferation assay.Briefly, 10⁴ suspension cells or 3×10³ SKBR3 cells were plated in 40 μlper well in a 96-well half-area white TC-treated plate and incubated at37° C. for 2 hours prior to adding antibodies. Antibodies wereformulated in the respective cell culture media and filter-sterilizedwith 0.45 μm celluloase aceteate Costar Spin-X®centrifuge tube filters(Corning; Tewksbury, Mass.). 40 μl of unconjugated antibody,AB4285-conjugated antibody or AB4285 free drug were added to cells andcultured for either 3 days or 5 days for suspension cells and SKBR3cells, respectively. Cell viability was measured by adding 80 μl of CellTiter-Glo™ reagent (Promega Corp.; Madison, Wis.) to each well andprocessed following product instructions. Luminescence was measured withthe ENVISION® plate reader (Perkin-Elmer; Waltham, Mass.). Relativeluminescence readings were converted to % viability using untreatedcells as controls and plotted versus antibody concentration. IC₅₀ wascalculated from the non-linear regression equation, log(inhibitor)versus response-variable slope (4 parameters) using the statisticalsoftware, Prism (GraphPad Software; San Diego, Calif.). Results areshown in FIGS. 8 and 9 and Table 14.

TABLE 14 Cell Killing by Brentuximab and Trastuzumab Variants Cell LineKarpas L540 SKBR3 IC50 IC50 IC50 ADC Site (nM) (nM) (nM) AglycosylatedBrentuximab HC F404 0.078 0.22 DBCO-MMAF 2 Aglycosylated Brentuximab HCK121 0.040 0.12 DBCO-MMAF 2 Aglycosylated Brentuximab LC N152 0.037 0.12DBCO-MMAF 2 Aglycoaylated Trastuzumab HC F404 0.043 DBCO-MMAF 2 freedrug DBCO-MMAF 2 243 646 378

Cell binding affinities of trastuzumab and brentuximab variants weremeasured using a FACS-based assay. Briefly, 2×10⁵ Karpas 299 or L540cells in 25 μl of binding buffer, which consists of PBS containing 0.5%bovine serum albumin (BSA) (Invitrogen; Carlsbad, Calif.) and 0.09%sodium azide (Sigma; St. Louis, Mo.) were plated in a 96-well U-bottompolypropylene plate (Greiner Bio-One; Monroe, N.C.). Antibodies wereformulated in binding buffer and 2-fold serial dilutions were preparedin a separate 96-well plate. Equal volume of diluted antibodies wereadded to cells and incubated at 4° C. for 1 hour. Cells were washedtwice with 200 μl of binding buffer to remove unbound antibodies. Forsecondary antibody detection, 5 μg/ml Alexa-488 conjugated goatanti-human IgG (Invitrogen; Carlsbad, Calif.) was prepared in bindingbuffer and 50 μl was added to cells and incubated at 4° C. for 1 hour.Cells were washed twice, resuspended in 200 μl of binding buffer andanalyzed using a BD FACScan® flow cytometer (Cytek; Fremont, Calif.)equipped with a 96-well automated microsampler (Cytek; Fremont, Calif.).Data was acquired and analyzed using FlowJo (Tree Star; Ashland, Oreg.).Mean fluorescence intensity readings were expressed as % binding bynormalizing to averaged maximum MFI values and data was plotted versusantibody concentration. Binding affinities was calculated from thenon-linear regression saturation binding equation, one site—totalbinding using Prism software. Results are shown in FIG. 10 and table 15.

TABLE 15 Cell Binding of Brentuximab Variants Cell Line Karpas L540 ADCSite Kd (nM) Kd (nM) Aglycosylated Brentuximab HC F404 0.46 0.81DBCO-MMAF 2 Aglycosylated Brentuximab HC K121 0.52 1.3 DBCO-MMAF 2Aglycosylated Brentuximab LC N152 0.50 1.0 DBCO-MMAF 2

Example 14 Antibody-Drug Conjugates in Alternative Scaffolds: scFvFormats

To demonstrate the feasibility of using scFv as alternative scaffold forADC, DNA encoding trastuzumab scFv (VL_VH) with an amber codon wascloned into expression vector pYD317. The TAG codon was inserted byoverlapping PCR mutagenesis at the nucleotides corresponding to theamino acid serine at the position (−1).

To express the scFv, cell-free extracts were thawed to room temperatureand incubated with 50 uM iodoacetamide for 30 min. Cell-free reactionswere run at 30 C for up to 10 h containing 30% (v/v)iodoacetamide-treated extract with 8 mM magnesium glutamate, 10 mMammonium glutamate, 130 mM potassium glutamate, 35 mM sodium pyruvate,1.2 mM AMP, 0.86 mM each of GMP, UMP, and CMP, 2 mM amino acids for all18 amino acids except tyrosine and phenylalanine which were added at 0.5mM, 4 mM sodium oxalate, 1 mM putrescine, 1.5 mM spermidine, 15 mMpotassium phosphate, 100 nM T7 RNAP, 1.3 uM E. coli DsbC, 2 mM oxidized(GSSG) glutathione, 10 ug/mL scFv plasmid 15 uM in vivo produced m.j.tRNA, 5 uM m.j RNA synthetase and 1 mM pAzido Phenylanine (pN3F). Wildtype scFv was expressed as control.

scFv(−1)pN3F and wild type scFv were purified by Protein L followed bySEC. 5 uM scFv (−1) was incubated with 50 uM of the DBCO-MMAF reagentshown in FIG. 1 for 16 hours at room temperature. The excess free drugwas removed by zeba desalting column.

Hydrophobic Interaction Chromatography was then performed to quantitatethe samples and to determine the drug-antibody ratios as follows.Samples and standards were diluted 1:1 in 3M Ammonium Sulfate (EMDChemical), 50 mM Sodium Phosphate pH 7.0 (Mallinckrodt) prepared inMilliQ water. A Dionex HPLC system was equipped with a Tosoh BioscienceLLC TSK-gel Butyl-NPR® (4.6 mm×3.5 cm) column with a column compartmenttemperature of 30° C. The mobile phase A was 1.5M Ammonium Sulfate, 50mM Sodium Phosphate, pH 7.0. The mobile phase B was 50 mM SodiumPhosphate, pH 7.0 in 80:20 water:isopropyl Alcohol (Honeywell). Themobile phase was delivered at a flow rate of 1.0 mL/minute. Theseparation was performed with a linear gradient of 15% mobile phase B to100% mobile phase B in 10 minutes. The UV data was acquired at 214 nm.The peak areas were quantitated using Chromeleon software (Thermo) tocalculate Drug Antibody Ratio (DAR).

To assess binding of the conjugated alternative scaffold variants, abinding assay to cells expressing HER2 was performed as follows. Thebinding of the purified conjugated variants to HER2 on SKBR3 cells,which overexpress the HER2/c-erb-2 gene product, with over 1.5 millionreceptor copies per cell (ATCC #HTB-30, Manassas, Va.) was compared toclinical grade Herceptin®, unglycosylated trastuzumab produced bycell-free protein synthesis, or human serum IgG1 as a negative control(Sigma-Aldrich; St. Louis, Mo.). SKBR3 cells were cultured in DMEM:Ham'sF-12 (50:50), high glucose (Cellgro-Mediatech; Manassas, Va.)supplemented with 10% heat-inactivated fetal bovine serum (Hyclone;Thermo Scientific; Waltham, Mass.), 2 mM glutamax (Invitrogen; Carlsbad,Calif.) and 1× Pencillin/streptomycin (Cellgro-Mediatech; Manassas,Va.). Adherent cells were washed twice with calcium and magnesium-freeHanks Balanced Salt Solution (HBSS), harvested with HYQ®TASE™ (Hyclone;Thermo Scientific; Waltham, Mass.). A total of 200,000 cells per samplein total volume of 10 μL were incubated with serial dilutions of eitherconjugated alternative scaffold variants, clinical grade HERCEPTIN®, oraglycosylated trastuzumab made in 10 μL FACS buffer (DPBS buffersupplemented with 1% bovine serum albumin). Cells plus antibody or ADCwere incubated for 60 minutes on ice. Unstained cells, human IgG1(Isotype control) and Secondary antibody (goat anti-human IgG) were usedas controls. To detect HERCEPTIN® or aglycosylated trastuzumab binding,cells were washed twice with ice-cold FACS buffer and incubated with 5g/ml Alexa 647 labeled goat anti-human IgG secondary antibody(Invitrogen; Carlsbad, Calif.) on ice for 1 hour. Alternative scaffoldvariant cell binding was detected by incubating cells on ice for 1 hwith either 5 g/ml Alexa 647 labeled Protein L (Pierce) or goatanti-human-Fc labeled with Alexa 647 (Invitrogen; Carlsbad, Calif.). Allsamples were washed using FACS buffer and analyzed using a BD FACSCalibur system (BD Biosciences; San Jose, Calif.).

Mean fluorescence intensities were fitted using non-linear regressionanalysis with one site specific binding equation using GraphPad Prism(GraphPad v 5.00, Software; San Diego, Calif.). Data was expressed asRelative MFI vs. concentration of antibody or antibody variant in nM.

Next, the effects of the conjugated alternative scaffold variants oncell killing were measured with a cell proliferation assay as follows.SKBR3 cells were obtained from ATCC and maintained in DMEM:Ham's F-12(50:50), high glucose (Cellgro-Mediatech; Manassas, Va.) supplementedwith 10% heat-inactivated fetal bovine serum (Hyclone; ThermoScientific; Waltham, Mass.), 2 mM glutamax (Invitrogen; Carlsbad,Calif.) and 1× Pencillin/streptomycin (Cellgro-Mediatech; Manassas,Va.). Adherent cells were washed twice with calcium and magnesium-freeHanks Balanced Salt Solution (HBSS), harvested with HYQ®TASE™ (Hyclone;Thermo Scientific; Waltham, Mass.). A total of 10³ cells were seeded ina volume of 401 in a 96-well half area flat bottom white Polystyreneplate. The cells were allowed to adhere overnight at 37° C. in a CO₂incubator. ADC variants were formulated at 2× concentration in DMEM/F12medium and filtered through MultiScreen _(HTS) 96-Well Filter Plates(Millipore; Billerica, Mass.). Filter sterilized conjugated alternativescaffold variants, HERCEPTIN®, or aglycoslyated trastuzumab were addedinto treatment wells and plates were cultured at 37° C. in a CO2incubator for 120 hrs. For cell viability measurement, 80 μl of CellTiter-Glo® reagent (Promega Corp.; Madison, Wis.) was added into eachwell, and plates processed as per product instructions. Relativeluminescence was measured on an ENVISION® plate reader (Perkin-Elmer;Waltham, Mass.). Relative luminescence readings were converted to %viability using untreated cells as controls. Data was fitted withnon-linear regression analysis, using log(inhibitor) vs.response-Variable slope, 4 parameter fit equation using GraphPad Prism(GraphPad v 5.00, Software; San Diego, Calif.). Data was expressed asrelative cell viability, ATP content % vs. dose of ADC in nM.

Results from the HIC analysis, the cell binding, and cell killingexperiments are presented in Table 16.

TABLE 16 scFv Cell Binding, Cell Killing, and DAR SKBR3 SKBR3 DAR byCell Binding Cell Killing HIC Scaffold IC50, nM Kd, nM AssayUnglycosylated Trastuzumab 5 NA NA scFv(−1) WT 15 NA NA scFv(−1)conjugate 14 0.48 0.77

Example 15 Antibody-Drug Conjugates in Alternative Scaffolds: scFv-FcFormats

To demonstrate the feasibility of using scFv Fc as alternative scaffoldfor ADC, DNA encoding trastuzumab scFv Fc (VL_VH_Fc) with amber wascloned into expression vector pYD317. TAG codon was inserted byoverlapping PCR mutagenesis at the nucleotides corresponding to theamino acid serine at the position (−1), Fc R355, Fc N389, and Fc F404(EU index numbering).

CFPS reaction conditions were same as described in Example 14, exceptthat 5 uM yeast PDI was added. Wild type scFv Fc was expressed ascontrol.

To conjugate aglycosylated scFv-Fc, proteins were first purified byProtein A followed by SEC. 5 uM pN3F containing scFv-Fc was incubatedwith 50 uM DBCO-MMAF for 16 hours at room temperature. The excess freedrug was removed by zeba desalting column.

Additionally, pAzido Methyl Phenylanine was incorporated to scFv Fc atthe sites of R355, N389, and F404, respectively. The reaction conditionwas same as described in Example 14, except that 1 uM m.j. pCyano FRSand 1 mM pAzido Methyl Phe were used to replace the pair of m.j. FRS andpAzdio Phe. The protein A purified protein was then conjugated withDBCO-MMAF reagent 2 (DBCO-MMAF 2). The structure of DBCO-MMAF 2 is shownin FIG. 11.

Next, LC-MS was performed to quantitate the samples and to determine thedrug-antibody ratios as follows. Samples were analyzed by liquidchromatography (CHIP-Cube nanoLC, Agilent) coupled to a qTOF massspectrometer (Agilent 6520). Proteins were separated on a reverse phaseHPLC-Chip (PLRP-S, 4000 Å, 5 g; Enrichment column: 25 mm; Separationcolumn: 150 mm×75 μm) with 0.1% formic acid in water (solvent A) and0.1% formic acid in acetonitrile+isopropyl alcohol (80:20 v/v, solventB). Data was processed using MassHunter Qualitative Software (Agilent).Data is shown in Table 17.

TABLE 17 DAR and Cell Binding and Killing of scFv-Fc variants SKBR3SKBR3 Cell Cell DAR by Binding Killing LCMS Scaffold Kd, nM IC50, nM DARHerceptin ® 12 NA NA Aglycosylated Trastuzumab 8.1 NA NA Her scFv-Fc 6.0NA NA scFv-Fc (−1) pN₃F DBCO-MMAF 6.8 0.011 1.86 scFv-Fc (R355) pN₃FDBCO-MMAF 6.9 0.015 1.77 scFv-Fc (N389) pN₃F DBCO-MMAF 5.4 0.010 1.53scFv-Fc (N389) pN₃CH₂F 3.0 0.052 1.97 DBCO-MMAF 2 scFv-Fc (R355) pN₃CH₂F 5.0 0.055 1.97 DBCO-MMAF 2 scFv-Fc (F404) vH-vL 0.05 1.99 pN3CH2 FDBCO-MMAF 2 scFv-Fc (F404) vL-vLH 0.068 1.97 pN3CH2 F DBCO-MMAF 2

In vivo stability of drug linker ADC conjugates was measured by dosing 2mg/kg of respective scFv-Fc DBCO-MMAF 2 conjugates into Beige nude Xidmice. Plasma was collected by terminal bleeds at 30 mins, d3, d7, d14and d21 from n=2 animals for each time point. Total circulating ScFv-FCADCs were captured by Biotin- (Fab)₂ Goat Anti-Human IgG, Fcγ fragmentspecific. Complex was pulled down with streptavidin Mag Sepharose.Captured complex was washed to remove nonspecific binding and elutedwith 1% formic acid. Neutralized eluate was analyzed by intact LCMS. Theresults indicate that scFv-Fc (N389) pN₃CH₂F DBCO-MMAF 2 and scFv-Fc(R355) pN₃CH₂ F DBCO-MMAF 2 are stable up to 28 days.

Next, pharmacokinetics properties of ScFv-Fc ADCs were analyzed in beigenude xid mice. Animals were dosed intravenously at a dose level ofapproximately of 2.0 mg/kg of scFv-Fc R355 and scFv-Fc N389. The plasmaconcentrations, sampled out to 28 days, were determined by immunoassayand the pharmacokinetic parameters calculated using a non-compartmentalapproach with WinNonlin ‘v’ 5.2 (Pharsight, Calif.). Results are shownin FIG. 12.

To assess in vivo efficacy of the scFc-Fc ADCs, KPL-4 human breast tumorcells were inoculated into the mammary fat pads of SCID beige mice(Charles River Laboratories). A total of 3 million cells per mouse,suspended in 50% phenol red-free Matrigel (Becton Dickinson Bioscience)mixed with culture medium were injected. Once tumor size was reached allanimals were randomly assigned into treatment groups, such that the meantumor volume for each group was 100-150 mm³.

Trastuzumab (30 mg/kg) was given i.p. (single injection on treatment day0), followed by (15 mg/kg) per week for 3 weeks. AglycosylatedTrastuzumab 2nnAA variant scFv-Fc N389 DBCO-MMAF 2 15 mg/kg (872 ugMMAF/m2, DAR 1.901), Aglycosylated Trastuzumab 2nnAA variant HC-S136 (15mg/kg) (unconjugated) Vehicle (PBS) and free drug (0.54 mg/kg) weregiven via i.v (single injection on treatment day 0). The results of thisexperiment are presented in FIG. 13 a.

In another experiment, scFv-Fc F404 vL-vH DBCO-MMAF 2 (15 mg/kg),scFv-Fc F404 vH-vL DBCO-MMAF 2 (15 mg/kg), Trastuzumab-CF HC F404DBCO-MMAF 2 (mg/kg), Trastuzumab-CF (15 mg/kg), and vehicle wereadministered by single i.v. injection on day 0. The results of thisexperiment are presented in FIG. 13 b.

For both experiments, all treatment groups consisted of 10 animals pergroup, and tumor size was monitored twice weekly using calipermeasurement. Mice were housed in standard rodent microisolator cages.Environmental controls for the animal rooms were set to maintain atemperature of 70° F., a relative humidity of 40% to 60%, and anapproximate 14-h light/10-h dark cycle.

The results of this experiment demonstrate that the ADCs conjugated atpositions N389 and F404 were effective in an animal to durably regresstumor size. Of particular note, the F404 scFv-Fc conjugates achievedregression below baseline, demonstrating particular efficacy for thisscaffold in a solid tumor model.

Example 16 Exemplary ADCs Specific for CD74 and Her2 and with AdditionalExemplary Linker-Warhead Combinations

This example provides exemplary antibody-drug conjugates prepared withan antibody specific for CD74 and containing different exemplarylinker-warhead combinations as described herein. This example furtherprovides exemplary antibody-drug conjugates prepared from trastuzumabconjugated with certain of the linker-warhead combinations.

The protein sequence of the heavy and light chains of the anti-CD74antibody used in this example are provided as SEQ ID NO:5 and 6,respectively. The heavy chain sequence includes a 6-His C-terminal tagto assist with puridication. Amber codons at the positions encoding HCK212, HC S136, HC F241, HC F404, LCS7, or LC N152 were introduced usingthe methods described in Example 13. Anti-CD74 antibodies containingp-methylazido-Phe were expressed and purified using the methodsdescribed in Example 13. Trastuzumab variants containingp-methylazido-Phe at positions HC S136 or HC F404 were also expressedand purified as described in Example 13.

Next, the anti-CD74 or trastuzumab variants were conjugated withDBCO-MMAF 2 (FIG. 11A), DBCO-DM4 (FIG. 11B), DBCO-DM4 2 (FIG. 11C), orDBCO-MMAE (FIG. 11D). The method used to perform the conjugationreactions between the antibody variants and the linker-warheadcombinations was as described in Example 13.

The drug-antibody ratios (DARs) of the ADCs was then determined by LC-MSaccording to the method described in Example 13. It should be noted thatthe LC-MS method for the anti-CD74 samples had not been optimized forthis antibody; the results of the analysis contained peaks overlappingwith unconjugated species, artificially lowering the DAR of thesesamples. The DAR of the ADC variants prepared in this example thereforeshowed good agreement with the ADCs containing a nnAA at correspondingsites discussed in Examples 1-15. Results of the DAR analysis arepresented in Table 18, below.

Next, the ADC variants were used in cell killing experiments todetermine their IC₅₀ values. Trastuzumab variants were analyzed asdescribed in Example 13. The effects of the conjugated anti-CD74variants on cell killing were measured by a cell proliferation assay.SU-DHL-6 and NCI-H929 cells were obtained from ATCC and maintained inRPMI-1640 medium (Cellgro-Mediatech; Manassas, Va.) supplemented with20% heat-inactivated fetal bovine serum (Hyclone; Thermo Scientific;Waltham, Mass.), 2 mM glutamax (Invitrogen; Carlsbad, Calif.) and 1×Pencillin/streptomycin (Cellgro-Mediatech; Manassas, Va.). SU-DHL-6 andNCI-H929 cells were harvested and re-suspended in culture medium atfinal concentration of 0.5×10⁶ cells/mL. A total of 20×10³ cells in avolume of 40 μl were seeded in each well of a 96-well half area flatbottom white polystyrene plate. ADC variants were formulated at 2×concentration in RPMI-1640 complete medium and filtered throughMultiScreen_(HTS) 96-Well Filter Plates (Millipore; Billerica, Mass.).Filter sterilized ADCs were added into treatment wells and plates werecultured at 37° C. in a CO₂ incubator for 72 hrs. For cell viabilitymeasurement, 80 μl of Cell Titer-Glo® reagent (Promega Corp. Madison,Wis.) was added into each well, and plates were processed as per productinstructions. Relative luminescence was measured on an ENVISION® platereader (Perkin-Elmer; Waltham, Mass.). Relative luminescence readingswere converted to % viability using untreated cells as controls. Datawas fitted with non-linear regression analysis, using log(inhibitor) vs.response-variable slope, 4 parameter fit equation using GraphPad Prism(GraphPad v 5.00, Software; San Diego, Calif.). Data was expressed asrelative cell viability, ATP content % vs. dose of ADC in nM. Results ofthe cell killing assays are presented in Table 18.

TABLE 18 DAR and IC₅₀ Values for Exemplary Anti-CD74 and Anti-HER2 ADCsAntibody Drug Conjugate Site DAR IC₅₀ Anti-CD74 Antibody DBCO-MMAF 2 HCK121 1.601 0.122 Anti-CD74 Antibody DBCO-MMAF 2 HC S136 1.601 0.100Anti-CD74 Antibody DBCO-MMAF 2 HC F241 1.58 0.078 Anti-CD74 AntibodyDBCO-MMAF 2 HC F404 1.631 0.137 Anti-CD74 Antibody DBCO-MMAF 2 LC S71.543 0.120 Anti-CD74 Antibody DBCO-MMAF 2 LC N152 1.457 0.130 Anti-CD74Antibody DBCO-DM4 HC K121 1.572 0.191 Anti-CD74 Antibody DBCO-DM4 HCS136 1.61 0.271 Anti-CD74 Antibody DBCO-DM4 HC F241 1.581 0.187Anti-CD74 Antibody DBCO-DM4 HC F404 1.531 0.253 Anti-CD74 AntibodyDBCO-DM4 LC S7 1.509 0.293 Anti-CD74 Antibody DBCO-DM4 LC N152 1.4740.281 Anti-CD74 Antibody DBCO-DM4 2 HC K121 1.578 0.215 Anti-CD74Antibody DBCO-DM4 2 HC S136 1.589 0.215 Anti-CD74 Antibody DBCO-DM4 2 HCF241 1.612 0.196 Anti-CD74 Antibody DBCO-DM4 2 HC F404 1.63 0.154Anti-CD74 Antibody DBCO-DM4 2 LC S7 1.561 0.291 Anti-CD74 AntibodyDBCO-DM4 2 LC N152 1.495 0.235 Anti-CD74 Antibody DBCO-MMAE HC K1211.811 0.166 Anti-CD74 Antibody DBCO-MMAE HC S136 1.752 0.242 Anti-CD74Antibody DBCO-MMAE HC F241 1.81 0.238 Anti-CD74 Antibody DBCO-MMAE HCF404 1.572 0.235 Anti-CD74 Antibody DBCO-MMAE LC S7 1.581 0.224Anti-CD74 Antibody DBCO-MMAE LC N152 1.616 0.251 Trastuzumab DBCO-DM4 HCF404 0.036 1.84 Trastuzumab DBCO-DM4 HC S136 0.038 1.82 TrastuzumabDBCO-DM4 2 HC F404 0.043 1.94 Trastuzumab DBCO-DM4 2 HC S136 0.038 1.82

Example 17 further demonstrates that the site of incorporation of a nnAAinto an antibody can transfer reasonably predictably between antibodies.Further, the identity of the linker and warhead does not appear tosignificantly affect the conjugation efficiency or DAR of the resultingADC, demonstrating reasonable predictability that a given warhead-linkercombination can be conjugated to the an antibody containing anon-natural amino acid at one of the preferred sites.

All publications and patent, applications cited in this specificationare herein incorporated by reference as if each individual publicationor patent application were specifically and individually indicated to beincorporated by reference. While the claimed subject matter has beendescribed in terms of various embodiments, the skilled artisan willappreciate that various modifications, substitutions, omissions, andchanges may be made without departing from the spirit thereof.Accordingly, it is intended that the scope of the subject matter limitedsolely by the scope of the following claims, including equivalentsthereof.

What is claimed is:
 1. An antibody fragment of the IgG class comprising an Fc protein, wherein the Fc protein comprises a polypeptide chain having a non-natural amino acid residue at heavy chain residue 404 according to the EU index of Kabat, or a post-translationally modified variant or an aglycosylated variant thereof, wherein the non-natural amino acid residue is selected from the group consisting of: ortho-substituted tyrosine, meta-substituted tyrosine, para-substituted phenylalanine, ortho-substituted phenylalanine, and meta-substituted phenylalanine.
 2. An antibody fragment of the IgG class comprising an Fc protein, wherein the Fc protein comprises a polypeptide chain having a non-natural amino acid at heavy chain residue 404 according to the EU index of Kabat, or a post-translationally modified variant or an aglycosylated variant thereof, wherein each non-natural amino acid residue is according to the formula

wherein each L is independently a divalent linker; each R is independently a reactive group, a therapeutic moiety or a labeling moiety, and wherein the non-natural amino acid residue is selected from the group consisting of: ortho-substituted tyrosine, meta-substituted tyrosine, para-substituted phenylalanine, ortho-substituted phenylalanine, and meta-sub stituted phenylalanine.
 3. The antibody fragment of claim 2 wherein each R is a reactive group selected from the group consisting of amino, carboxy, acetyl, hydrazino, hydrazido, semicarbazido, sulfanyl, azido and alkynyl.
 4. The antibody fragment of claim 2 wherein each L is a divalent linker selected from the group consisting of a bond, alkylene, substituted alkylene, heteroalkylene, substituted heteroalkylene, arylene, substituted arylene, heteroarlyene and substituted heteroarylene.
 5. The antibody fragment of claim 1, wherein the Fc protein is aglycosylated.
 6. The antibody fragment of claim 5, wherein the Fc protein has a higher thermal stability (T_(m)1) compared to the corresponding wild-type Fc protein.
 7. A composition comprising the antibody fragment of claim 5, wherein said composition is substantially pure.
 8. A composition comprising the antibody fragment of claim 5 wherein said antibody fragment is at least 95% by mass of the total protein mass of said composition.
 9. The antibody fragment of claim 5, wherein the Fc protein does not comprise a variable domain or a light chain.
 10. The antibody fragment of claim 5, wherein the Fc protein corresponds to a subclass selected from the group consisting of Ig1, IgG2, IgG3, and IgG4.
 11. The antibody fragment of claim 5, wherein said non-natural amino acid residue comprises a moiety selected from the group consisting of amino, carboxy, acetyl, hydrazino, hydrazido, semicarbazido, sulfanyl, azido and alkynyl.
 12. The antibody fragment of claim 5, wherein each non-natural amino acid residue is according to the formula

wherein each L is independently a phenylene; and each R is independently a functional group selected from the group consisting of a therapeutic moiety, a labeling moiety, amino, carboxy, acetyl, hydrazino, hydrazido, semicarbazido, sulfanyl, azido and alkynyl.
 13. The antibody fragment of claim 5, wherein the non-natural amino acid residue is selected from the group consisting of: p-acetyl-L-phenylalanine, O-methyl-L-tyrosine, 3-methyl-phenylalanine, O-4-allyl-L-tyrosine, 4-propyl-L-tyrosine, fluorinated phenylalanine, isopropyl-L-phenylalanine, p-azido-L-phenylalanine, p-acyl-L-phenylalanine, p-benzoyl-L-phenylalanine, p-iodo-phenylalanine, p-bromophenylalanine, p-amino-L-phenylalanine, isopropyl-L-phenylalanine, p-propargyloxy-phenylalanine, and p-azidomethyl-L-phenylalanine.
 14. The antibody fragment of claim 5, wherein the non-natural amino acid residue is p-azido-L-phenylalanine.
 15. The antibody fragment of claim 5, wherein the non-natural amino acid residue is p-azidomethyl-L-phenylalanine.
 16. An Fc protein conjugate comprising the antibody fragment of claim 5, wherein the Fc protein is linked to one or more therapeutic moieties or labeling moieties.
 17. The Fc protein conjugate of claim 16, wherein the Fc protein is linked to one or more drugs or polymers.
 18. The Fc protein conjugate of claim 16, wherein the Fc protein is linked to one or more labeling moieties.
 19. The Fc protein conjugate of claim 16, wherein said Fc protein is linked to one or more single chain binding domains (scFv).
 20. The Fc protein conjugate of claim 16, wherein said one or more therapeutic moieties or labeling moieties is linked to the non-natural amino acid, or a residue thereof, via one or more linkers. 